KR20060053824A - Drug composition comprising nf-kappa; b inhibitor - Google Patents

Abstract

A drug composition for ameliorating the symptoms attributed to tumor cells, comprising a compound represented by the general formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient. General formula: (1) wherein R1 represents hydrogen or a C2-4 alkanoyl group; and R2 represents a group of any of the following formulae: (A) - (G) in which R3 represents a C1-4 alkyl group.

Description

Pharmaceutical composition containing NF-κＢ inhibitors {DRUG COMPOSITION COMPRISING NF-κB INHIBITOR}             

Cross reference with related literature

This application claims priority based on Japanese Patent Application No. 2002-185866, filed June 26, 2002, and Japanese Patent Application No. 2003-37167, filed February 14,2003. These documents are incorporated in this specification.

The present invention relates to pharmaceutical compositions, tumor cell proliferation inhibitors, adhesion molecule expression inhibitors, apoptosis inducers, arteriosclerosis or cancer prevention and treatment agents, cachexia treatment agents, and treatment methods.

In recent years, homeostasis of NF-κB has been reported successively in various tumors. For example, bladder cancer (Hum. Gene Ther. 10: 37-47, 1999), breast cancer (Cancer Res. 9: 3810-3818, 2001), melanoma (Cancer Res. 61: 4901-4949, 2001), etc. NF-κB is frequently activated in tumors. Such activation of NF-κB is likely to inactivate apoptosis-inducing function and promote tumor progression. This is also suggested by inhibiting NF-κB by high expression of IκB and inducing cell death specifically for neoplastic cells having high NF-κB activity (Hum. Gene Ther. 10: 37-47, 1999). ).

In addition, cachexia (also referred to as villain) is an anorexia, progressive weight loss, anemia, dry skin, edema, etc. in chronic diseases such as malignant tumor, tuberculosis, diabetes, blood disease, endocrine and metabolic diseases. The main symptom is systemic defects. Especially in terminal patients of malignant tumors and the like, the symptoms often appear, indicating a decrease in systemic function mainly on weight loss and anemia. When cancer patients develop cachexia, the risk of complications increases and the response to chemotherapy worsens. In addition, due to systemic weakness, side effects of chemotherapy and radiation therapy for cancer are also increased, and death may occur due to cachexia.

Until now, the detailed mechanism by which cachexia develops is not fully elucidated, but in recent years has involved involvement of several cytokines such as interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α). At last, a clue about the mechanism has been obtained (Latest Medicine 1999 No. 54, No. 10, pp. 2502-2507). For example, as a mechanism of expression of various symptoms in cancer cachexia, cytokines overexpressed and induced by cachexia act on the central nervous system, causing symptoms such as decreased eating, fever, hypotension, and lethargy, It also leads to a hypertrophy of sugar, protein and lipid catabolism.

Steroid administration is effective in suppressing symptoms in cachexia. When steroids are administered to cachexia patients, metabolic abnormalities of cachexia are corrected by suppressing the immune response of the steroids, anti-inflammatory action thereof, and inhibiting the production of cachexia-induced cytokines, thereby reducing weight, anorexia, lethargy, Symptoms of cachexia such as abnormal taste and anemia are alleviated and improved. However, the long-term use of steroids is a serious problem. Since steroids are originally hormones of their own, ingested steroids have the same effects as excess hormones. For example, steroids may be involved in resorption of salts in the kidneys, resulting in edema and hypertension.

On the other hand, administration of eicosapentaenoic acid (EPA) has a certain effect on the improvement of cachexia by using omega-3 unsaturated fatty acids that inhibit the production of inflammatory cytokines such as IL-6 or affect the synthesis of acute phase reactive proteins. It is raising. However, since such a nutritional agent is indirect in action, a remarkable and definite effect is hardly expected.

Therefore, there is a need for development of a drug that is different from a drug having a wide range of actions such as steroids, is specific for cachexia, and has a significant effect. Under such a request, for example, since salidamide has an inhibitory effect of TNF-α, improvement of cachexia symptoms is expected, and therefore, it has been used as a treatment for cancer cachexia. However, since TNF-α has another action in vivo called angiogenesis, administration of salidemide causes side effects of inhibiting angiogenesis. As described above, even if the drug is relatively high in specificity, side effects always occur. Therefore, in consideration of use under various circumstances, development of various drugs having different mechanisms of action is desired.

On the other hand, mevarotin and the like are used for atherosclerosis, which reduces cholesterol and indirectly has a therapeutic effect, but the effect is insufficient. There is no clinical use as a cancer cell metastasis inhibitor. Metal protease inhibitors and the like are under development, but this is not a promise. Common anticancer drugs have a strong side effect, and it is well known that their use is significantly limited.

Then, an object of this invention is to provide the pharmaceutical composition which can improve the symptom accompanying activation of NF-κB.

NF-κB is a transcription factor that functions in the nucleus, but in the presence of its endogenous inhibitor, IκB, they form a complex and exist in the cytoplasm as an inactive form. When cells are stimulated by TNF-α or the like, the degradation of IκB is induced and NF-κB is activated. The activated NF-κB migrates into the nucleus and binds to the NF-κB binding site of DNA, resulting in an immune response or inflammation. Genes encoding cytokines involved in the reaction (eg, IL-1, IL-2, IL-8, TNF-α, etc.) and cell adhesion molecules (eg, ICAM-1, VCAM-1, etc.) (Ghoshi, S. et al., Annu. Rev. Immunol. 16: 225-260 (1998)). As such, one of the intracellular target molecules for the expression of TNF-α is thought to be NF-κB.

In recent years, as a substance having an activation inhibitory effect of NF-κB, a compound represented by the following formula (1) has been developed (International Publication No. WO 01/12588 Pamphlet; Matsumoto et al., Bioorg.Med.Chem. Lett. 10, 865 (2000).

(In formula, R <^1> is a hydrogen atom or a C2-C4 alkanoyl group, and R <^2> is group represented by either of following formula (A)-(G).)

(In formula, R <^3> is a C1-4 alkyl group.)

Since atherosclerosis and cancer cell metastasis require expression of adhesion factors of vascular endothelial cells, the present inventors solved the problem by using a drug that does not express adhesion factors. Similarly, if IL-6 and TNF-α are involved in the mechanism of cachexia development, it is considered that the impairment of NF-κB, the intracellular target molecule, is effective for the prevention / improvement of symptoms caused by cachexia. .

Therefore, when the compound was administered to the model mouse that caused cachexia symptoms and the symptoms were observed, it was found that it was effective for the prevention / improvement of cachexia symptoms, thereby completing the present invention. In the present specification, the symptom refers to a wide range of phenomena caused by the disease, and does not necessarily indicate only the apparent abnormality that the patient complains of.

That is, the pharmaceutical composition according to the present invention is characterized by containing a compound represented by the following formula (1) or a pharmacologically acceptable salt thereof as an active ingredient for improving at least one symptom caused by tumor cells. .

[Formula 1]

In formula, R <^1> is a hydrogen atom or C2-C4 alkanoyl group, As an alkanoyl group, an acetyl, propionyl, butanoyl group, and these isomer groups are mentioned, Especially an acetyl group is preferable.

R <^2> is group represented by either of following formula (A)-(G).

In formula, R <^3> is a C1-C4 alkyl group, As an alkyl group, a methyl group, an ethyl group, a propyl group, a butyl group, and these isomer groups are mentioned, Especially a methyl group and an ethyl group are preferable.

In addition, at least one symptom may be improved by the apoptosis of the tumor cells, and the apoptosis of the tumor cells does not contribute, and at least one symptom resulting from the tumor cells is improved. It may be characterized by.

In addition, by inhibiting NF-κB activation, at least one symptom resulting from the tumor cells may be improved.

The symptom is, for example, tumor metastasis. In addition, tumor metastasis may be improved by inhibiting adhesion with vascular endothelial cells.

By inhibiting the proliferation of the tumor cells, at least one symptom resulting from the tumor cells may be improved.

The above symptom is, for example, a symptom selected from the group consisting of Hodgkin's disease, cancer cachexia and leukemia. The tumor cells are, for example, breast cancer cells.

Moreover, the said compound may be characterized by following formula (1a) or (1b).

At least one symptom may be prevented or improved among the weight loss, the decrease in hematocrit value, the decrease in fat, and the decrease in muscle, which are symptoms of the cancer cachexia. However, all the symptoms associated with cachexia are not limited to these, and even if the skin is dry or edema, it belongs to the technical scope of the present invention.

In addition, the pharmaceutical composition according to the present invention may be characterized by improving at least one symptom caused by the tumor cells by inhibiting angiogenesis in the tumor formed by the tumor cells.

Moreover, the pharmaceutical composition which concerns on this invention is a compound represented by following General formula (1) which can augment the said therapeutic effect by inhibiting the said activation of NF-κB by the treatment which activates NF-κB, or its pharmacological agent It is characterized by containing an acceptable salt as an active ingredient.

[Formula 1]

In formula, R <^1> is a hydrogen atom or C2-C4 alkanoyl group, As an alkanoyl group, an acetyl, propionyl, butanoyl group, and these isomer groups are mentioned, Especially an acetyl group is preferable.

R <^2> is group represented by either of following formula (A)-(G).

In formula, R <^3> is a C1-C4 alkyl group, As an alkyl group, a methyl group, an ethyl group, a propyl group, a butyl group, and these isomer groups are mentioned, Especially a methyl group and an ethyl group are preferable.

The treatment for activating the NF-κB may be a treatment using an anti-tumor agent, or may be a treatment by radiation of tumor cells. Furthermore, you may make it contain the said pharmaceutical composition and the said anti-tumor agent as an active ingredient. The antitumor agent is, for example, camptothecin or daunorbicin.

Moreover, the said compound may be characterized by following formula (1a) or (1b).

A tumor cell proliferation inhibitor for inhibiting the proliferation of tumor cells according to the present invention is characterized by containing a compound represented by the following formula (1) or a pharmacologically acceptable salt thereof as an active ingredient.

[Formula 1]

In formula, R <^1> is a hydrogen atom or the alkanoyl group of C2-C4, As an alkanoyl group, an acetyl, propionyl, butanoyl group, and these isomer groups are mentioned, Especially an acetyl group is preferable.

R <^2> is group represented by either of following formula (A)-(G).

In formula, R <^3> is a C1-C4 alkyl group, A methyl group, an ethyl group, a propyl group, a butyl group, and these isomer groups are mentioned as an alkyl group, A methyl group and an ethyl group are especially preferable.

Moreover, the said compound may be characterized by following formula (1a) or (1b).

The adhesion molecule expression inhibitor for inhibiting the expression of adhesion molecules of vascular endothelial cells according to the present invention is characterized by containing a compound represented by the following formula (1) or a pharmacologically acceptable salt thereof as an active ingredient.

[Formula 1]

In formula, R <^1> is a hydrogen atom or the alkanoyl group of C2-C4, As an alkanoyl group, an acetyl, propionyl, butanoyl group, and these isomer groups are mentioned, Especially an acetyl group is preferable.

R <^2> is group represented by either of following formula (A)-(G).

In formula, R <^3> is a C1-C4 alkyl group, A methyl group, an ethyl group, a propyl group, a butyl group, and these isomer groups are mentioned as an alkyl group, A methyl group and an ethyl group are especially preferable.

Moreover, the said compound may be characterized by following formula (1a) or (1b).

An apoptosis inducer for inducing apoptosis of tumor cells according to the present invention is characterized by containing a compound represented by the following formula (1) or a pharmacologically acceptable salt thereof as an active ingredient.

[Formula 1]

In formula, R <^1> is a hydrogen atom or the alkanoyl group of C2-C4, As an alkanoyl group, an acetyl, propionyl, butanoyl group, and these isomer groups are mentioned, Especially an acetyl group is preferable.

R <^2> is group represented by either of following formula (A)-(G).

In formula, R <^3> is a C1-C4 alkyl group, A methyl group, an ethyl group, a propyl group, a butyl group, and these isomer groups are mentioned as an alkyl group, A methyl group and an ethyl group are especially preferable.

The said compound may be characterized by following formula (1a) or (1b).

The agent for preventing and treating atherosclerosis of the present invention is characterized by containing a compound having an NF-κB inhibitory activity as an active ingredient. The compound having the NF-κB inhibitory effect may be a compound represented by the following formula (1) or a pharmacologically acceptable salt thereof.

[Formula 1]

In formula, R <^1> is a hydrogen atom or the alkanoyl group of C2-C4, As an alkanoyl group, an acetyl, propionyl, butanoyl group, and these isomer groups are mentioned, Especially an acetyl group is preferable.

R <^2> is group represented by either of following formula (A)-(G).

In formula, R <^3> is a C1-C4 alkyl group, A methyl group, an ethyl group, a propyl group, a butyl group, and these isomer groups are mentioned as an alkyl group, A methyl group and an ethyl group are especially preferable.

The preventive and therapeutic agent for cancer of the present invention is characterized by containing a compound having an NF-κB inhibitory activity as an active ingredient. The compound having an NF-κB inhibitory effect may be a compound represented by the following formula (1) or a pharmacologically acceptable salt thereof.

[Formula 1]

In formula, R <^1> is a hydrogen atom or the alkanoyl group of C2-C4, As an alkanoyl group, an acetyl, propionyl, butanoyl group, and these isomer groups are mentioned, Especially an acetyl group is preferable.

R <^2> is group represented by either of following formula (A)-(G).

In formula, R <^3> is a C1-C4 alkyl group, A methyl group, an ethyl group, a propyl group, a butyl group, and these isomer groups are mentioned as an alkyl group, A methyl group and an ethyl group are especially preferable.

The prophylactic or therapeutic agent may be used for inhibiting metastasis of cancer.

The cachexia therapeutic agent of the present invention is characterized by containing a compound represented by the following formula (1) or a pharmacologically acceptable salt thereof as an active ingredient.

[Formula 1]

In formula, R <^1> is a hydrogen atom or the alkanoyl group of C2-C4, As an alkanoyl group, an acetyl, propionyl, butanoyl group, and these isomer groups are mentioned, Especially an acetyl group is preferable.

R <^2> is group represented by either of following formula (A)-(G).

In formula, R <^3> is a C1-C4 alkyl group, A methyl group, an ethyl group, a propyl group, a butyl group, and these isomer groups are mentioned as an alkyl group, A methyl group and an ethyl group are especially preferable.

Moreover, the said compound may be characterized by following formula (1a) or (1b).

Moreover, these compounds may be characterized by being a treatment for cancer cachexia in tumor patients (eg, gallbladder cancer patients), but if the patient is developing cachexia, the cause may not be cancer.

In the case of tumor patients (eg, gallbladder cancer patients), at least one symptom is prevented or improved among weight loss, hematocrit reduction, fat loss, and muscle reduction, which are symptoms of cancer cachexia. You may make it. However, all the symptoms associated with cachexia are not limited to these, and even if the skin is dry or edema, it belongs to the technical scope of the present invention.

Furthermore, the cachexia therapeutic agent of the present invention may contain a compound having an NF-κB inhibitory activity as an active ingredient.

The treatment method according to the present invention is characterized by using a compound represented by the following formula (1) or a pharmacologically acceptable salt thereof for improving at least one symptom caused by tumor cells.

[Formula 1]

In formula, R <^1> is a hydrogen atom or the alkanoyl group of C2-C4, As an alkanoyl group, an acetyl, propionyl, butanoyl group, and these isomer groups are mentioned, Especially an acetyl group is preferable.

R <^2> is group represented by either of following formula (A)-(G).

In formula, R <^3> is a C1-C4 alkyl group, A methyl group, an ethyl group, a propyl group, a butyl group, and these isomer groups are mentioned as an alkyl group, A methyl group and an ethyl group are especially preferable. Further, the treatment method includes a prevention method, a progression inhibiting method, and the like.

At least one symptom may be improved by the apoptosis of the tumor cells, and the at least one symptom resulting from the tumor cells may be improved without apoptosis of the tumor cells. You may make it. In addition, by inhibiting NF-κB activation, at least one symptom resulting from the tumor cells may be improved.

The symptoms are, for example, a tumor metastasis, a symptom resulting from the proliferation of the tumor cells, Hodgkin's disease, a cancerous fluid, a symptom selected from the group consisting of.

Moreover, the said compound may be characterized by following formula (1a) or (1b).

The therapeutic method according to the present invention is characterized by using a compound represented by the following formula (1) or a pharmacologically acceptable salt thereof for improving arteriosclerosis by inhibiting adhesion between vascular endothelial cells and leukocytes. It is done.

[Formula 1]

In formula, R <^1> is a hydrogen atom or the alkanoyl group of C2-C4, As an alkanoyl group, an acetyl, propionyl, butanoyl group, and these isomer groups are mentioned, Especially an acetyl group is preferable.

R <^2> is group represented by either of following formula (A)-(G).

In formula, R <^3> is a C1-C4 alkyl group, A methyl group, an ethyl group, a propyl group, a butyl group, and these isomer groups are mentioned as an alkyl group, A methyl group and an ethyl group are especially preferable. Further, the treatment method includes a method of preventing arteriosclerosis, a method of inhibiting progression of arteriosclerosis, and the like.

Moreover, the said compound may be characterized by following formula (1a) or (1b).

In addition, the therapeutic method according to the present invention comprises the steps of performing a treatment for activating NF-κB, and administering a pharmaceutical composition containing a compound represented by the following formula (1) or a pharmacologically acceptable salt thereof as an active ingredient: It characterized by including a step to make.

[Formula 1]

In formula, R <^1> is a hydrogen atom or the alkanoyl group of C2-C4, As an alkanoyl group, an acetyl, propionyl, butanoyl group, and these isomer groups are mentioned, Especially an acetyl group is preferable.

R <^2> is group represented by either of following formula (A)-(G).

In formula, R <^3> is a C1-C4 alkyl group, A methyl group, an ethyl group, a propyl group, a butyl group, and these isomer groups are mentioned as an alkyl group, A methyl group and an ethyl group are especially preferable. Furthermore, the treatment method includes a prevention method, a progression suppression method, and the like.

The treatment for activating the NF-κB may be administration of an anti-tumor agent or treatment by radiation of tumor cells.

Moreover, the said compound may be characterized by following formula (1a) or (1b).

BRIEF DESCRIPTION OF THE DRAWINGS It is a schematic diagram explaining the mechanism which the compound which has an NF-κB inhibitory effect suppresses arteriosclerosis and metastasis of cancer.

2 is a diagram showing the inhibitory effect by DHMEQ when stimulated with TNF-α.

Fig. 3 is a diagram showing the effect of DHMEQ inhibiting the expression of ICAM-1, VCAM-1, and E-selectin when stimulated with TNF-α.

4 is a diagram showing the effect of DHMEQ inhibiting adhesion of HUVECs and leukocytes (top) or HUVECs and HL-60 cells (bottom).

Fig. 5 is a diagram showing the results of analysis of gel shift assays on the inhibitory effect of DHM2EQ on constitutive NF-κB activation of ATL cells.

6 is a diagram showing the results of analysis of a reporter gene assay regarding the inhibitory effect of DHM2EQ on constitutive NF-κB activation of ATL cells.

Fig. 7 is a diagram showing the results of analysis of confocal microscopy on the inhibitory effect of DHM2EQ on constitutive NF-κB activation of ATL cells.

Fig. 8 is a graph showing the results of concentration dependency analysis of proliferation inhibitory effect on DTL A2 cells of DHM2EQ.

9 is a diagram showing the results of the time-lapse analysis of the proliferation inhibitory effect of DHM2EQ on ATL cells.

Fig. 10 is a diagram showing an analysis result of proliferation inhibitory effect of DHM2EQ on peripheral blood cells of ATL patients.

Fig. 11 is a diagram showing an analysis result of proliferation inhibitory effect on normal peripheral blood monocytes of DHM2EQ.

12 is a diagram showing an analysis result of apoptosis inducing action of DHM2EQ on ATL cells.

Fig. 13 is a diagram showing an analysis result of proliferation inhibitory effect on Hodgkin's lymphoma cells of DHM2EQ.

14 is a diagram showing an analysis result of proliferation inhibitory effect on multiple myeloma cells of DHM2EQ.

Fig. 15 is a diagram showing the effect of DHMEQ inhibiting the activation of NF-κB induced by TNF-α.

Fig. 16 shows the proliferation inhibitory effect of DHMEQ on MCF-7.

Fig. 17 is a diagram showing the proliferation inhibitory effect of DHMEQ on SCID mouse transplanted human breast cancer cell line MCF-7.

Fig. 18 is a graph showing the proliferation inhibitory effect of Lewis lung tumor and COX-2 inhibitor celecoxib on HT-29.

19 is a graph showing luciferase activity when transfecting p6kb-Luc to JCA-1 cells and administering various concentrations of DHMEQ in one embodiment of the present invention.

20 is a graph showing the temporal change of body weight of mice when DHMEQ was administered to gallbladder cancer mice inoculated with JCA-1 cells in one embodiment of the present invention.

FIG. 21 is a graph showing the temporal change in tumor weight calculated from tumor diameter when DHMEQ is administered to gallbladder cancer mice inoculated with JCA-1 cells in one embodiment of the present invention.

22 is a graph showing the tumor weight on day 26 after the start of DHMEQ administration to gallbladder cancer mice inoculated with JCA-1 cells in one embodiment of the present invention.

FIG. 23 is a graph showing the weight of fat around the testis on day 26 after initiation of DHMEQ in gallbladder cancer mice inoculated with JCA-1 cells in one embodiment of the present invention.

24 is a graph showing the weight of gastrocnemius muscle at 26 days after the start of DHMEQ administration to gallbladder cancer mice inoculated with JCA-1 cells in one embodiment of the present invention.

25 is a graph showing hematocrit values 26 days after the start of DHMEQ administration to gallbladder cancer mice inoculated with JCA-1 cells in one embodiment of the present invention.

FIG. 26 is a table showing weights of organs measured by dissecting 26 days after the start of DHMEQ administration to gallbladder cancer mice inoculated with JCA-1 cells in one embodiment of the present invention. FIG.

27 is a diagram showing the results of constitutive NF-κB activation inhibitory activity of multiple myeloma (MM) cell line by DHMEQ in one embodiment of the present invention.

28 is a diagram showing the results of the proliferation inhibitory action of multiple myeloma (MM) cell line by DHMEQ in one embodiment of the present invention.

29 is a diagram showing the results of the proliferation inhibitory action of multiple myeloma (MM) patient cells by DHMEQ in one embodiment of the present invention.

30 is a diagram showing the results of constitutive NF-κB activation inhibitory activity of Hodgkin's lymphoma (HL) cell line by DHMEQ in one embodiment of the present invention.

Figure 31, in one embodiment of the present invention, is a figure showing the results of the antitumor agent enhancement effect by DHMEQ.

32 is a diagram showing that in one embodiment of the present invention, the antitumor activity enhancing effect by DHMEQ is due to inhibition of NF-κB activation by the antitumor agent.

Fig. 33 is a diagram showing the results of adjusting the effect of DHMEQ in vivo using SCID mice inoculated with ATL cell line intraperitoneally in one embodiment of the present invention.

34 is a diagram showing the results of the investigation of the apoptosis enhancing effect of DHMEQ in radiation-treated tumor cells in one embodiment of the present invention.

35 is a diagram showing the results of investigating the proliferation inhibitory effect of tumor cells on human pancreatic cancer of DHMEQ in vivo in one embodiment of the present invention.

36 is a diagram showing the results of the investigation of the in vitro proliferation inhibitory effect on the human pancreatic cancer cell line in the radiation combination of DHMEQ in one embodiment of the present invention.

The objects, features, advantages and ideas of the present invention will be apparent to those skilled in the art by the description herein. Embodiments of the invention, specific examples, and the like described below show preferred embodiments of the invention and are shown for the purpose of illustration or description, and the invention is not limited thereto. It is apparent to those skilled in the art that various modifications and variations are possible within the scope and spirit of the present invention disclosed herein, based on the description described herein.

The pharmaceutical composition according to the present invention can improve at least one symptom resulting from tumor cells. To ameliorate the symptoms caused by the tumor cells, apoptosis may be generated in the tumor cells. Examples of the target symptom of this method include Hodgkin's disease and leukemia, but the present invention is not limited thereto. In addition, the apoptosis of the tumor cells does not have to contribute to the improvement of the symptoms caused by the tumor cells. Examples include, but are not limited to, tumor metastasis, cancer cachexia, and the like. Here, the fact that the apoptosis of tumor cells does not contribute means that even if the pharmaceutical composition of the present invention is administered to the affected area, the effect does not depend on the apoptosis of the tumor cells in the affected area, Apart from the non-dependent effects, it is possible to have apoptosis in tumor cells. In addition, a tumor has the same meaning as a cancer of a broad range, for example, it refers to a lymphoid malignant tumor, breast cancer, a gallbladder cancer, pancreatic cancer, etc.

Activation of NF-κB has been reported to be involved in various aspects of tumorigenesis, including control of apoptosis, cell proliferation, cell differentiation (Albert S. Baldwin, J. Clin. Invest. 107: 241). -246 (2001). Furthermore, activation of NF-κB in cancer cells by chemotherapy or radiotherapy has been reported to reduce the effectiveness of cancer treatment (Albert S. Baldwin, ex.). Therefore, the present inventors designed the structure of the antibiotic epoxyquinomycin C (epoxyquinomicin C) in a circle as described above, and the synthesized DHMEQ represented by the above-mentioned formula (1) has anticancer activity. It is feed.

When the inventors investigated the effect on the activation of NF-κB in adult T cell leukemia lymphoma (ATL) cells using DHMEQ, it became clear that DHMEQ inhibited the activation of NF-κB in ATL cells. In addition, when examining the effect on the proliferation of ATL cells using DHMEQ, it became clear that DHMEQ did not inhibit the proliferation of normal cells but inhibited the proliferation of ATL cells. Further, when examining the effect on the induction of apoptosis of ATL cells, it became clear that DHMEQ induces apoptosis of ATL cells but not apoptosis of normal cells. Further, examples of compounds having an NF-κB inhibitory activity include salicylate amide derivatives (WO01 / 12588 A1), panepoxydone (Biochem. Biophys. Res. Commun. 226, 214-221, 1996), cycloepoxydone (cycloepoxydon; J. Antibiot. 51, 455-463, 1998), SN-50 (J. Biol. Chem. 270, 14255-14258). Other methods for producing compounds having NF-κB inhibitory activity are described in the following documents. Panepoxydone is available from Biochem. Biophys. Res. Commun. 226, 214-221, 1996 and cycloepoxydone is described in J. Antibiot. 51, 455-463, 1998, SN50 is described in J. Biol. Chem. 270, 14255-14258, 1995.

In addition, when the inventors investigated the effects on the proliferation of Hodgkin's lymphoma cells using DHMEQ, the DHMEQ inhibits the proliferation of Hodgkin's lymphoma cells activated by NF-κB, but the NF-κB is not activated. It was clear that the proliferation of leukemia cells of the myeloid line was not inhibited. In addition, when the effects on the proliferation of multiple myeloma cells were examined using DHMEQ, it became clear that DHMEQ also inhibited the proliferation of multiple myeloma cells.

Conventional chemotherapy targets a widespread growth mechanism that is common to cells, and thus has a large effect on normal cells as well as tumor cells, and is not necessarily an effective treatment. However, the NF-κB inhibitor DHMEQ represented by the above formula (1) induces apoptosis of leukemia cells on which NF-κB is activated and does not induce apoptosis of human normal leukocytes at the same concentration. From the experimental results (FIGS. 8 and 11), it was clear that the specificity for the tumor cells was high. Therefore, DHMEQ has fewer side effects than conventional chemotherapy and is more useful as a pharmaceutical composition for tumors starting with malignant tumors.                 

DHMEQ is also useful as an apoptosis inducer of tumor cells. For example, the pharmaceutical composition according to the present invention can prevent or treat lymphoid malignant tumors by inhibiting the proliferation of lymphoid malignant tumor cells and inducing apoptosis of lymphoid malignant tumor cells. The type of lymphoid malignancy to be prevented or treated is not particularly limited, but may be suitably used for the prevention or treatment of malignant lymphoma, leukemia or myeloma. Malignant lymphomas include non-Hodgkin's lymphoma, Hodgkin's lymphoma, and the like, and myeloma includes plasmacytic tumors such as multiple myeloma and the like. Etc. are included.

The tumor cell proliferation inhibitor according to the present invention can inhibit the proliferation of lymphoid malignant tumor cells by the proliferation inhibitory effect of lymphoid malignant tumor cells, and thereby prevent or treat lymphoid malignant tumors. The type of lymphoid malignant tumor cells to be inhibited for proliferation is not particularly limited, but may be suitably used for inhibiting the proliferation of malignant lymphoma cells, leukemia cells or myeloma cells. Malignant lymphoma, leukemia or myeloma include various lymphoid malignancies exemplified above.

The apoptosis inducer according to the present invention can induce apoptosis of lymphoid malignant tumor cells by apoptosis inducing action of lymphoid malignant tumor cells, thereby preventing or treating lymphoid malignant tumors. . The type of lymphoid malignant tumor cells to be subjected to apoptosis is not particularly limited, but can be suitably used for inducing apoptosis of malignant lymphoma cells, leukemia cells or myeloma cells. Malignant lymphoma, leukemia or myeloma include various lymphoid malignancies as exemplified above.

Since the pharmaceutical composition, tumor cell proliferation inhibitor, and apoptosis inducer according to the present invention act specifically on lymphoid malignant tumor cells and show little adverse effects on normal cells, excellent prevention and treatment of lymphoid malignancies is achieved. You can expect the effect.

The compound represented by the formula (1) (particularly DHMEQ) and its pharmacologically acceptable salts also act on cancer cells, but act on the epilepsy of cancer tissue (part of cancer tissue composed of normal cells). In particular, in vascular endothelial cells in cancer tissues, DHMEQ does not cause apoptosis, but suppresses the expression of adhesion factors and the like (FIG. 5), thereby providing an inhibitory effect on cancer progression.

When inflammatory stimuli or physical stimuli reach the vascular endothelial cells, the expression of adhesion molecules is enhanced, and white blood cells adhere to the surface of vascular endothelial cells and migrate out of the blood vessel. This is because NF-κB, a transcription factor in vascular endothelial cells, is activated, thereby activating the expression of adhesion molecules such as ICAM-1, VCAM-1, and E-selectin. In addition, high-metastatic colorectal cancer has been reported to have high expression of cyl Lewis-X, a ligand of E-selectin. When NF-κB, a transcription factor in vascular endothelial cells, is activated, colon cancer cells appear on the surface of vascular endothelial cells. It is considered that it is easy to adhere to the surface, thereby leaching out of blood vessels, and acts as a facilitating transition. Therefore, it is thought that inhibiting the expression of the adhesion molecules on the surface of vascular endothelial cells leads to inhibition of arteriosclerosis and tumor metastasis, and NF-κB inhibitors are considered useful for inhibiting arteriosclerosis and metastasis of cancer cells.                 

Thus, the present inventors investigated the effect of DHMEQ on adhesion molecule expression of human umbilical vein endothelial cells (HUVECs) using DHMEQ. When the activation of NF-κB by TNF-α was evaluated by gel shift assay, the activation of NF-κB was suppressed without inhibiting the degradation of IκB-α. In addition, when the effects of DHMEQ on the expression of ICAM-1, VCAM-1, and E-selectin induced by TNF-α in Western blotting were examined, the expression of these adhesion molecules was suppressed, and the adhesion of leukocytes and HUVECs was actually observed. They found that they also inhibited the adhesion of leukemia cells to HUVECs. The adhesion of leukocytes to the blood vessel wall induces atherosclerosis through the accumulation of lipids and the like, while the adhesion of cancer cells causes leaching and metastasis in blood vessels. Thus, inhibiting these adhesions leads to anti-arterial hardeners in the former and cancer metastasis inhibitors in the latter. Therefore, the compound having NF-κB inhibitory activity is useful for inhibiting atherosclerosis or metastasis of cancer cells (FIG. 1). Moreover, according to the Example mentioned later, DHMEQ is useful as an inhibitor of expression of the adhesion factor of vascular endothelial cells.

Another example of the apoptotic independence effect is cell proliferation inhibition. The inventors of the present invention, when using DHMEQ to investigate the effect on human breast cancer cells in vitro and in vivo proliferation, it was clear that DHMEQ inhibits the proliferation of human breast cancer cells. The compound represented by general formula (1) and its pharmacologically acceptable salt have the effect of inhibiting the proliferation of breast cancer cells. Therefore, such a compound is useful as an agent for preventing and treating breast cancer.

Conventionally, two types of treatment of breast cancer are known, chemotherapy (treatment using an anticancer agent) and hormone therapy. All of these methods are known to have an effect of reducing cancer and preventing recurrence, but there are also problems. That is, in the case of anticancer drugs, there is the biggest problem of toxicity (side effects), and drug resistance was also a big problem. In hormonal therapy, there is a problem in that it is effective only for hormone-sensitive cancer. Hormone-sensitive cancer is about 60% of the total, and the remaining 40% of patients cannot apply hormone therapy from the outset. In addition, hormonal therapy has emerged as a resistance, which is a big problem. Therefore, DHMEQ is extremely useful for breast cancer.

In addition, the present inventors are concerned that IL-6 and TNF-α are involved in the mechanism of cachexia, which is frequently seen in chronic diseases, especially in terminal patients with malignant tumors, and thus, impairs the function of NF-κB, an intracellular target molecule. In addition, it was considered effective for the prevention / improvement of symptoms caused by cachexia and investigated whether DHMEQ is useful for cachexia. That is, when DHMEQ was administered to the model mouse that caused cachexia symptoms and the symptoms were observed, it was found that DHMEQ is effective for preventing / improvement of cachexia symptoms. This made it clear that DHMEQ is also useful for cachexia.

In addition, since the pharmaceutical composition according to the present invention can inhibit the activation of NF-κB, it is possible to suppress the gene expression of cyclooxygenase-2 (COX-2) caused by the activation of NF-κB. do. Moreover, since gene expression of cyclooxygenase-2 can be suppressed, the synthesis | combination of prostaglandin can also be suppressed. It is thought that this can inhibit or inhibit the neovascularization of tumor vessels promoted by prostaglandins. Therefore, the pharmaceutical composition according to the present invention is expected to be useful as a cancer therapeutic agent that exhibits antitumor activity by inhibiting angiogenesis in the tumor and inhibiting oxygen or nutrient supply to the tumor.

In addition, the present inventors think that the effect of tumor treatment may be increased by inhibiting the activation of NF-κB in tumor cells by treatments that activate NF-κB such as chemotherapy or radiotherapy. First, the effect of enhancing the antitumor activity by DHMEQ was investigated. In other words, when antitumor activity enhancement effect by DHMEQ was examined using antitumor agents such as camptothecin (CPT) and daunomycin (DNR), DHMEQ enhances the effects of antitumor agents. Was found.

Moreover, the present inventors investigated the NF-κB activation by each antitumor agent treatment. When treated with any antitumor agent (camptothecin (CPT), daunomycin (DNR), etoposide (ETP)), 3 to 20-fold NF-κB activity was routinely applied to tumor cells compared to that before treatment. I found something that was springing up. As a result, it was clear that the antitumor effect enhancing effect by DHMEQ was due to inhibition of NF-κB activation by the antitumor agent.

Next, in order to investigate inhibition of NF-κB activation by antitumor agents by DHMEQ, tumor cells were treated by using DHMEQ in combination with each antitumor agent (camptothecin (CPT) and daunomycin (DNR)). When examining NF-κB activation at one time, it was found that NF-κB activation was strongly inhibited by the combination of DHMEQ even if treated with any antitumor agent.

On the other hand, radiation therapy generates active enzymes, which may damage the DNA of normal tissues, causing other cancers or worsening the remaining cancer cells. In addition, when NF-κB activation is induced by oxidative stress caused by radiation, cancer cells are less likely to cause apoptosis and are known to be resistant to radiation therapy. Therefore, the present inventors considered that synergistic effects were obtained even when DHMEQ and radiation were used in combination, and investigated the in vitro proliferation inhibitory effect on tumor cells in combination with radiation of DHMEQ. As a result, it was found that DHMEQ exhibits a synergistic inhibitory effect in combination with irradiation.

As described above, the pharmaceutical composition according to the present invention is also useful as an inhibitor of NF-κB activation caused by treatment with an anti-tumor agent or treatment by irradiation with tumor cells. Moreover, the pharmaceutical composition which concerns on this invention is useful also in combination with an antiviral agent. Furthermore, the treatment using an antitumor agent is not particularly limited as long as it is an antitumor agent that activates NF-κB, and examples thereof include camptothecin and daunovirin.

The pharmaceutical composition according to the present invention can increase the effect of treatment by using in combination with the treatment used for diseases such as tumor diseases, allergic diseases, immune diseases, inflammatory diseases and the like. Although the pharmaceutical composition of this invention may be used simultaneously with the treatment which activates NF-κB, it may be used in order to inhibit the activation of NF-κB after the treatment which activates NF-κB. Moreover, in order to inhibit the activation of NF-κB, you may administer beforehand to the person with the said disease, and a mammalian animal other than a human.

On the other hand, one of the major problems of current anticancer drug treatment is the frequent occurrence of drug-resistant drug resistance to cancer cells. Actions on normal cells generally make it difficult to develop resistance. Therefore, this is an advantage of the prophylactic and therapeutic agent for breast cancer of the present invention. Next, low toxicity is expected, which is an advantage when compared with chemotherapy. Furthermore, from the mechanism of action, hormonal insensitive tumors that are not targeted by hormonal therapy are also subject to treatment. This is a big advantage compared to hormone therapy. As mentioned above, the low toxicity, the thing which does not overlap with a hormonal therapy, and the hard to produce tolerance to a vascular endothelial cell etc. are a clinical advantage. Moreover, such a point may suggest the possibility of using together with the existing chemical | medical agent widely, and application to cancer prevention.

From the above, the pharmaceutical composition according to the present invention includes tumor cell proliferation inhibitors, adhesion molecule expression inhibitors, apoptosis inducers, arteriosclerosis or cancer prevention / treatment agents, lymphocytic tumor prevention / treatment agents, breast cancer prevention / treatment agents, It can be said that it is useful for cachexia treatment.

Therefore, a pharmaceutical composition containing the compound represented by the formula (1) according to the present invention as an active ingredient is useful for the treatment (including prevention and progression inhibitors) of tumors, cachexia, arteriosclerosis, and the like.

Hereinafter, the manufacturing method, use form, and use example (example) of the compound of this invention are described in detail.

=== Method for Preparing Compound of the Present Invention ===

The compound represented by General formula (1) can be manufactured according to the synthesis method (Synthesis, No. 12, 1549-1561, 1995), such as Wipf.                 

Below, an example of the manufacturing method of the compound represented by General formula (1) is demonstrated based on the following reaction process formula.

Step a: Preparation of N- (2-alkanoylbenzoyl) -2,5-dimethoxyaniline

Ethyl acetate of O-alkanoylsaltyloyl halide of formula (7) is dissolved in 2,5-dimethoxyaniline in a solvent (pyridine or the like) and -78 to 50 ° C, preferably ice-cooling. A solution is added and reacted with stirring. After stopping the reaction by adding water, ethyl acetate was added, washed sequentially with hydrochloric acid, water, sodium bicarbonate water and water, and after drying, concentrated under reduced pressure and vacuum drying, to form N- (2-alkanoyl). A benzoyl) -2,5-dimethoxyaniline compound is obtained. This compound can be used in the next step without purification.                 

Step b: Preparation of 3- (O-alkanoylsaltyloylamide) -4, 4-dialkoxy-2,5-cyclohexadienone compound

The compound of the formula (2) obtained above is dissolved in a solvent such as methanol, and diacetoxy iodine benzene is added at -20 to 50 ° C, preferably under ice cooling, and the reaction is stirred at room temperature. After concentration under reduced pressure, ethyl acetate was added, the mixture was washed sequentially with sodium bicarbonate and brine, and the residue obtained by concentrating the solvent under reduced pressure was purified by column chromatography, thereby obtaining 3- (O-alkanoylsaltylo) represented by the formula (3). Monoamide) -4,4-dialkoxy-2,5-cyclohexadienone compound.

Step c: preparation of 5, 6-epoxy-4,4-diakoxy-3-saltyloylamide-2-cyclohexenone compound

3- (O-alkanoylsaltyloylamide) -4,4-dialkoxy-2 and 5-cyclohexadienone represented by the formula (3) are dissolved in a solvent (tetrahydrofuran, methanol, etc.), Hydrogen peroxide and sodium hydroxide are added under -20-50 degreeC, Preferably it is ice-cooling, and it reacts with stirring. Ethyl acetate is added to the reaction solution, the mixture is washed sequentially with a hydrochloric acid solution, an aqueous sodium thiosulfate solution, and brine, dried in air, and then dried in vacuo. In order to remove the remaining raw material compound, the residue is dissolved in acetone, p-toluenesulfonic acid is added and stirred at room temperature to decompose the raw material compound. Ethyl acetate is added to the residue obtained by distilling off methanol under reduced pressure, and the mixture is washed with water. The residue obtained by drying the ethyl acetate layer was purified by column chromatography to obtain 5, 6-epoxy-4, 4-diakoxy-3-saltyloylamide-2-cyclohexenone represented by the formula (4). Obtain the compound.                 

Step d: Preparation of 5, 6-epoxy-2-saltyloylamide-2-cyclohexene-1, 4-dione

5,6-Epoxy-4,4-diakoxy-3-saltyloylamide-2-cyclohexenone compound of the formula (4) is dissolved in a solvent (methylene chloride, etc.), and an inorganic acid or an organic acid Boron fluoride diethyl ether complex, etc.) is added and reacted with stirring. A solvent (such as ethyl acetate) was added to the reaction solution, washed with water, the ethyl acetate layer was concentrated, and the obtained residue was washed with methanol to obtain 5,6-epoxy-2-saltyloyl represented by the formula (5). Obtain amide-2-cyclohexene-1,4-dione.

Step e: preparation of 5,6-epoxy-4-hydroxy-3-saltyloylamide-2-cyclohexenone (1a, DHM2EQ)

5, 6-epoxy-2-saltyloylamide-2-cyclohexene-1,4-dione represented by the formula (5) is suspended in a solvent (methanol, ethanol, THF, etc.), and -78 to 50 The reaction is carried out by adding a reducing agent (such as sodium borohydride) at 0 ° C, preferably under ice-cooling. A solvent (ethyl acetate, methylene chloride, etc.) was added to the reaction solution, and the mixture was washed sequentially with hydrochloric acid and water. The solvent layer was dried, concentrated under reduced pressure, suspended with methanol, stirred and washed to obtain 5, represented by the formula (1a). 6-Epoxy-4-hydroxy-3-saltyloylamide-2-cyclohexenone (DHM2EQ) is obtained.

Step f: Preparation of 3,3-dialkoxy-4, 5-epoxy-6-hydroxy-2-saltyloylamide cyclohexene

5, 6-epoxy-4, 4-dialkoxy-3-saltyloylamide-2-cyclohexenone compound of the formula (4) is dissolved in a solvent such as methanol and a mixed solution of sodium bicarbonate, -78 to 50 A reducing agent (such as sodium borohydride) is added to the reaction mixture under stirring, preferably at ice cooling. A solvent (such as ethyl acetate) was added to the reaction solution, and the mixture was washed sequentially with hydrochloric acid and water. The solvent layer was dried, concentrated under reduced pressure, dried in vacuo, purified by column chromatography, and the like (3) and (3). -Dialkoxy-4, 5-epoxy-6-hydroxy-2-saltyloylamide cyclohexene is obtained.

Step g: Preparation of 5, 6-epoxy-4-hydroxy-2-saltyloylamide-2-cyclohexenone (1b, DHM3EQ)

3, 3-dialkoxy-4, 5-epoxy-6-hydroxy-2-saltyloylamide cyclohexene represented by the formula (6) is dissolved in a solvent (such as acetone) to form p-toluenesulfonic acid. It is added and reacted with stirring at room temperature. A solvent (such as ethyl acetate) was added to the reaction solution, washed with water, the solvent layer was dried, concentrated under reduced pressure, and purified to give 5, 6-epoxy-4-hydroxy-2-saltylo of formula (1b). Obtain amide-2-cyclohexenone (DHM3EQ).

The compound represented by the formula (1) is a weakly acidic substance, and salts thereof include salts of organic bases such as quaternary ammonium salts, or salts with various metals such as alkali metals such as sodium. And also in the form of these salts. These salts can be manufactured by a well-known method.

= = = Modes of Use of Compounds of the Invention = = =

 The compound represented by the formula (1) or a pharmacologically acceptable salt thereof may be used alone or in combination with another drug (for example, another anticancer agent or hormone therapy agent).                 

In the case of administering a compound represented by the formula (1) or a pharmacologically acceptable salt thereof to a human, for example, about 1 to 100 mg / kg (body weight) per day, preferably 4 to 12 mg / kg (body weight) The dose or frequency of administration may be divided into one or several doses, but the dose and the number of times of administration may vary depending on the type of disease, symptoms, age, weight, duration of treatment, treatment effect, and method of administration. It can change suitably according to the above.

The compound represented by the formula (1) or a pharmacologically acceptable salt thereof may be combined with a pharmacologically acceptable carrier and orally administered as an agent such as an emulsion, a tablet, a capsule, a granule, a powder, a syrup, or the like. As a preparation, it is injected subcutaneously, intramuscularly, intraperitoneally or intravenously, or rectally, as a suppository (suppository), or sprayed into the oral or airway mucosa as a preparation such as a spray, or an ointment or tape It is also possible to administer parenterally in an aspect such as applying or affixing to an affected part (for example, skin or mucous membrane) as a preparation of.

Liquid preparations such as emulsions and syrups include sugars such as water, sucrose, sorbitol and fructose, glycols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil and soybean oil, and preservatives such as p-hydroxybenzoic acid esters. Flavors such as strawberry flavored peppermint and peppermint may be prepared as additives. Capsules, tablets, powders, granules, etc. include excipients such as lactose, glucose, sucrose, mannitol, starch, disintegrating agents such as sodium arginate, glidants such as magnesium stearate and tar, polyvinyl alcohol, Binders, such as hydroxypropyl cellulose and gelatin, surfactants, such as fatty acid ester, and plasticizers, such as glycerin, can be manufactured as an additive.                 

Injectables can be prepared, for example, using a salt solution, a glucose solution or a mixture thereof as a carrier. Suppositories can be produced using, for example, cacao oil, hydrogenated fats or carboxylic acids as a carrier. The spraying agent can be prepared using lactose, glycerin, etc. as a carrier which does not irritate the oral cavity and airway mucosa of the recipient, and also facilitates absorption by dispersing the active ingredient into fine particles. It can be formulated with a dry powder or the like.

As a pharmacologically acceptable carrier, one or two or more kinds of conventional organic or inorganic carrier materials may be used as the formulation material and formulated. Specifically, water, a pharmacologically acceptable organic solvent, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium arginate, water soluble dextran, carboxymethyl starch sodium, pectin, chiantan gum, Arabia Rubber, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose and the like.

As the additive used in the formulation, for example, in solids, excipients, lubricants, binders, disintegrants, liquids, solvents, dissolution aids, suspending agents, isotonic agents, buffers, analgesics, etc. Also good. If necessary, additives such as preservatives, antioxidants, colorants, sweeteners, fillers, extenders, dehumidifying agents, surfactants, stabilizers, bactericides, chelating agents, pH adjusters, and surfactants may be used. These additives are appropriately selected depending on the dosage unit form of the preparation and the like. Among these additives, components used in conventional formulations, for example, stabilizers, fungicides, buffers, isotonic agents, chelating agents, pH adjusters, surfactants and the like are preferably selected.

Specific examples of the additives are illustrated below.

Stabilizer: human serum albumin; L-amino acids, such as glycine, cysteine, and glutamic acid; Monosaccharides such as glucose, mannose, galactose and fructose, sugar alcohols such as mannitol, inositol and xylitol, disaccharides such as sucrose, maltose and lactose, polysaccharides such as dextran, hydroxypropyl starch, chondroitin sulfate and hyaluronic acid and Sugars such as derivatives thereof; Cellulose derivatives such as methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose and sodium carboxymethyl cellulose.

Surfactant: Surfactant, such as a polyoxyethylene glycol sorbitan alkyl ester, a polyoxyethylene alkyl ether type, a sorbitan monoacyl ester type, and a fatty acid glyceride type.

Buffers: boric acid, phosphoric acid, acetic acid, citric acid, ε-aminocaproic acid, glutamic acid and salts thereof (e.g. alkali metal salts or alkaline earth metal salts such as sodium salts, potassium salts, calcium salts and magnesium salts).

Isotonic agents: sodium chloride, potassium chloride, sugars, glycerin, etc.

Chelating Agents: Sodium Edetate, Citric Acid, etc.

The content rate of the compound represented by General formula (1) or its pharmacologically acceptable salt (active ingredient) in a formulation can be fluctuated between 1 to 90 weight%. For example, when taking the form of a tablet, a capsule, a granule, a powder, etc., it is preferable to contain 5 to 80 weight% of active ingredients. In the case of liquid preparations, such as a syrup, it is preferable to contain 1-30 weight% of active ingredients. Furthermore, in the case of injection administered parenterally, it is preferable to contain 1-10 weight% of active ingredients.

Formulation of the compound represented by the formula (1) or a pharmacologically acceptable salt thereof includes excipients (sugars such as lactose, white sugar, glucose, mannitol, starch such as potato, wheat, corn, calcium carbonate, calcium sulfate, sodium hydrogen carbonate) Inorganic materials such as crystalline cellulose), binders (starch paste solution, gum arabic, gelatin, sodium arginate, methyl cellulose, ethyl cellulose, polyvinylpyrrolidone, polyvinyl alcohol, hydroxypropyl cellulose, carmellose, etc.), glidants Agent (magnesium stearate, tar, hydrogenated vegetable oil, macrogol, silicone oil), disintegrant (starch, agar, gelatin powder, crystalline cellulose, CMCNa), CMC Ca, calcium carbonate, sodium bicarbonate, sodium arginate Etc.), copper colloid (lactose, white sugar, glucose, mannitol, aromatic oils, etc.), solvents (injectable water, sterilized purified water, sesame oil, soybean oil, corn oil, olive oil, cottonseed oil, etc.), stabilizers (nitrogen, carbon dioxide) Inert gas, EDTA, chelating agents such as thioglycolic acid, sodium bisulfite, sodium thiosulfate, L-ascorbic acid, reducing substances such as longalit, etc.), preservatives (paraoxybenzoic acid ester, chlorobutanol, benzyl) Alcohols, phenols, benzalkonium chloride, etc.), surfactants (hydrogenated himashi oil, polysorbate 80, 20, etc.), buffers (citric acid, acetic acid, sodium salt of phosphoric acid, boric acid, etc.), diluents, etc. It is done by the method.

EMBODIMENT OF THE INVENTION Hereinafter, the Example which concerns on this invention is described in detail. In the following description, unless otherwise specified, J. Sambrook, EF, Fritsch & T. Manatis (Ed.), Molecular cloning, a laboratory manual (3rd edition), Cold Spring Harbor Press, Cold Spring Harbor, New York ( 2001); F.M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, K. Struhl (Ed.), Current Protocols in Molecular Biology, John Wiley & Sons Ltd. The method described in the standard protocol collection, etc., or the method which modified or modified this is used. In addition, when using a commercially available reagent kit or measuring apparatus, unless there is a description in particular, the attached protocol is used for these.

In addition, the compound (DHMEQ) represented by General formula (1a) used by the following example was manufactured by the method of Examples 1-5 of WO01 / 12588 A1. Hereinafter, the compound represented by general formula (1a) may be called "DHM2EQ", and the compound represented by general formula (1b) may be called "DHM3EQ".

<Example 1>

When examining the activation of NF-κB by a gel shift assay (EMSA), DHMEQ produced TNF-α (Techne), in HUVECs (prepared from the umbilical cord at Keio Hospital). Activation of NF-κB by IL-1β (PEPRO TECH EC LTD) and LPS (Sigma) was completely inhibited to 3 μg / ml. 2 shows the inhibitory effect on NF-κB activation by DHMEQ when HUVECs were stimulated with TNF-α. The experimental method is as follows.

HUVECs were pretreated with DHMEQ as follows. That is, DHMEQ diluted with methanol was incubated at 37 ° C. and 5% CO ₂ for 2 hours by adding 1% of the amount of the culture solution so as to have the concentration indicated in the data in the culture solution incubating HUVECs.

HUVECs treated with DHMEQ for 2 hours and untreated HUVECs were stimulated with 10 ng / ml TNF-α, 10 ng / ml IL-1β, and 10 ug / ml LPS, respectively (i.e., added to each culture and labeled with data). Time (0, 5, 10, 30 minutes, etc.), 37 ° C, incubated with 5% CO ₂ ), and the cells were sampled. The collected cells were suspended in 400 μl of buffer A (10 mM HEPES (Sigma): pH 7.9, 1.5 mM DTT (Merck), 0.1 mM PMSF (Sigma)), and left to stand for 15 minutes to 15000 × g. The supernatant was removed by centrifugation for 5 minutes. Again 400 μl of buffer A was added and centrifuged at 15000 × g for 5 minutes to remove supernatant. Next, 40 μl of buffer C (20 mM HEPES: pH7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl ₂ , 0.2 mM DTT, 0.2 mM PMSF) was added thereto. The cells were suspended by tapping with a finger, allowed to stand for 20 minutes, and then centrifuged at 15000 x g for 5 minutes to recover the supernatant, which was used as a nuclear extract. 4 μl of the following synthetic oligonucleotide (Promega), 2 μl of [γ- ³² P] -ATP (Amersham), 2 μl of 10 × T4 PNK buffer (0.5M Tris-HCl (Sigma): pH7.6, 0.1 mM MgCl ₂ , 50 mM DTT, 1 mM spermidine.HCl (Sigma), and 1 mM EDTA were added and the total amount was 18 µl with distilled water. Further, 2 µl of 10 units / µl of T4 polynucleotide kinase (Takara) was added and reacted at 37 ° C for 10 minutes. After the reaction, 80 µl of TE buffer (10 mM Tris-HCl: pH7.5, 1 mM EDTA) was added to stop the reaction. This was purified by Nick column (Amersham) and an NF-κB probe labeled ³² P was obtained.

Synthetic Oligonucleotides:

5'-AGT TGA GGG GAC TTT CCC AGG C-3 '(SEQ ID NO: 1)

3'-TCA ACT CCC CTG AAA GGG TCC G-5 '(SEQ ID NO: 2)

Next, 5 × binding buffer (375 mM NaCl, 75 mM Tris-HCl: pH7.0, 7.5 mM EDTA, 7.5 mM DTT, 37.5% glycerol, 1.5% NP-40 (Nakaraitesk), 0.5 μg / ml BSA (Sigma) 1 μl and 5 μg of poly dI-dC (Amersham) adjusted to 4 μl and 1 μg / μl) were added, and distilled water was added so that the total amount was 17 μl. Samples for super shift were also added with 0.5 μg of anti p65 antibody (Santa Cruz). 3 µl of an NF-κB probe labeled ³² P was added thereto and incubated at 25 ° C for 30 minutes. 4% polyacrylamide gel (6.7 mM acrylamide; 30/2; Wah), 1.25 ml 10 × TBE buffer (0.9M Tris-boric acid) ): pH 20.3, 20 mM EDTA), 42 ml H ₂ O, 500 μl ammonium peroxodisulfate (APS), 50 μl TEMED (applied) wells were applied to the 20 μl sample and run at 150 V. did. After electrophoresis, the gel was transferred to filter paper and dried using a gel dryer, which was then exposed to a film.

<Example 2>

When the effects of DHMEQ on the expression of ICAM-1, VCAM-1, and E-selectin when stimulated with TNF-α by Western blotting were examined, TNF-α alone The expressions of ICAM-1 and VCAM-1 peak at 9 hours after stimulation, and the expression of E-selectin peaks at 6 hours after stimulation, but as shown in FIG. 3, HUVECs pretreated with DHMEQ for 2 hours are shown in FIG. Expression of any of the adhesion molecules was also significantly suppressed. The experimental method is as follows.

HUVECs pretreated with DHMEQ for 2 hours and untreated HUVECs were stimulated with 10 ng / ml TNF-α, respectively, and cells were sampled. It contains lysis buffer (20mM Tris-HCl: pH 8.0, 150mM NaCl, 2mM EDTA, 100mM NaF, 400μM Na ₃ VO ₄ , 1% NP-40, 1μg / ml Leupeptin (Microbial chemical research institute), 1 µg / ml antipain (research microbial chemical research institute), 1 mM PMSF) were added, solubilized for 30 minutes with stirring every 5 minutes on ice, and then to 15000 x g. The supernatant was recovered by centrifugation for 10 minutes. Protein concentration of the supernatant was quantified by Coomassie Brilliant Blue Liquid (Bio-Rad), and after adjusting the concentration, 3 × SDS loading buffer (150 mM Tris-HCl: pH 6.8, 30% glycerol, 3% SDS (關 東) Chemistry), 0.03 mg / ml bromophenol blue, and 150 µl / ml 2-mercaptoethanol (2-mercaptoethanol) were added in half the amount of the added lysis buffer, and the mixture was boiled for 5 minutes. This was taken as a sample and electrophoresed using a 12.5% polyacrylamide gel. After electrophoresis, the protein in the gel was transferred to a PVDF membrane (Amersham) and blocked with TBS buffer (20 mM Tris-HCl: pH 7.6, 137 mM NaCl) containing 5% skim milk. Subsequently, antibodies of each of ICAM-1 (Santa Cruz), VCAM-1 (Santa Cruz), and E-selectin (Santa Cruz) were used for antibody reaction with the PVDF membrane, and further, the appropriate secondary antibody (ICAM-). 1, VCAM-1, and secondary antibodies of E-selectin (Amersham and Santa Cruz)). Then, color development was carried out by the ECL method and the film was exposed to light.

<Example 3>

As shown in FIG. 4, leukocytes (prepared from the blood of the experimenter) and HL-60 cells (purchased from the cell bank) were stimulated with TNF-α, and cell adhesion after 3, 6, and 9 hours increased in time-dependent manner, but DHMEQ When 3 µg / ml was treated, the adhesion was remarkably suppressed. This concentration of DHMEQ showed no toxicity and proliferation inhibition against HUVECs. The experimental method is as follows.

HUVECs were aliquoted into 24 well plates and incubated to confluent. The composition of the medium was 9.8 g / l medium 199 (donated from Nisui), 1.8 g / l NaHCO ₃ (WaKo), 10 ml / l 1M Hepes buffer (Sigma), 30 mg / l ECGS (Becton Dickinson), 6 ml / l Heparin sodium injection solution, 10% heat-inactivated FBS (JRH). Then, the following experiment was done. HUVECs pretreated with DHMEQ for 2 hours and untreated HUVECs were stimulated with 10ng / ml TNF-α, respectively, and HUVECs from HL-60 cells of 0, 3, 6 and 9 hours post-stimulation and human acute promyeloid leukemia lines The adhesion to was evaluated. This time, monocytes isolated by specific centrifugation were used as white blood cells.

After stimulation with TNF-α, the wells were washed twice with HBSS + (donated from Nisui) and 500 μl of medium (composition of medium: 9.8 g / l medium 199 (donated from Nisui)), 1.8 g / l NaHCO ₃ (Wako), 10 ml / l 1 M Hepes buffer (Sigma), 30 mg / l ECGS (Becton Dickinson), 6 ml / l heparin sodium injection solution, 10% heat-inactivated FBS (JRH), Leukocytes were aliquoted into wells at 2.5 × 10 ⁵ cells / well and HL-60 cells at 7 × 10 ⁴ cells / well. Incubated for 1 hour at 37 ° C. with 5% CO ₂ , then washed gently three times with HBSS +, and photographed to count the number of cells adhered to HUVECs.

As described above, the DHMEQ is useful for long-term use in experiments and clinical trials because it inhibits the activation of NF-κB that does not exhibit toxicity and proliferation inhibition and inhibits adhesion of HUVECs with leukocytes and leukemia cells. It is feed.

<Example 4>

Inhibitory Effect of DHM2EQ on Constitutive NF-κB Activation in Adult T Cell Leukemia Lymphoma (ATL) Cells

(1) Gelshift Essay

The following method examined whether the transcription factor present in the nucleus of the cell binds to the region having the specific nucleotide sequence of the promoter.

2 × 10 ⁶ ATL cell lines (MT-1, TL-0ml) and control cell lines (K562, Jurkat + TNF) were treated with 10 μg / ml of DHM2EQ for 12 hours to prepare a nuclear extract from each cell line. In addition, nuclear extracts were prepared in the same manner from each cell line not treated with DHM2EQ. Furthermore, cell lines MT-1 and TL-0ml are cell lines transformed with HTLV-1, and NF-κB is activated in these cell lines. Cell line K562 (myeloid cell leukemia cell line) is a cell line that does not recognize constitutive NF-κB (negative control), and Jurkat + TNF is a Jurkat cell line treated with TNF (positive control).

NF-κB consensus oligomer (Promega) was end-labeled using [γ- ³² P] -ATP and polynucleotide kinase (PNK) to construct a ³² P labeled NF-κB probe.

2 μg of the protein amount of the nuclear extract and 10,000 μm of the NF-κB probe were mixed at a volume of 20 μl and allowed to react at room temperature for 30 minutes. The solution after the reaction was electrophoresed in a 7.5% polyacrylamide gel, and the gel was dried and then exposed to an X-ray film.

The results are shown in FIG. In the figure, in the six lanes from the left, "-" represents the result of DHM2EQ unprocessed, and "+" represents the result of DHM2EQ processing. In the positive control of the right two lanes, "-" and "+" are the results of contention inhibition ("+" is experiment in the presence of contention molecule) by the unlabeled probe, as shown to "comp" and the right side. Indicates that the signal is specific for the NF-κB binding sequence.

As shown in FIG. 5, the NF-κB signal was almost lost in the ATL cell lines (MT-1, TL-0ml) by DHM2EQ treatment. On the other hand, activation of NF-κB was not observed in K562.

(2) Reporter gene assay (Luciferase assay)

To examine the transcriptional activity of NF-κB, a luciferase construct (6κB-Luc plasmid) driven by an artificial promoter having 6 copies of NF-κB binding sequence was used as a reporter, and the plasmid DNA was used as a reporter. Transient was introduced into ATL cell lines (MT-1, TL-0ml) and control cell line (K562) (transfected using DMRIE-C (INVITROGEN), on a scale of 2 × 10 ⁵ cells / transfection). ). After 12 hours, the cells were recovered with 5 μg / ml of DHM2EQ, and after 48 hours, cells were recovered, and the transcriptional activity of NF-κB was evaluated by the enzyme activity of luciferase. Moreover, the same evaluation was performed also in each cell line which was not processed by DHM2EQ. Further, the entire experiment was conducted three times to calculate the mean value and standard deviation.

The results are shown in FIG. In addition, in the figure, "-" shows the result of DHM2EQ non-processing, and "+" shows the result of DHM2EQ process.

As shown in FIG. 6, in ATL cell lines (MT-1, TL-0ml), luciferase activity was inhibited to about 50% by DHM2EQ treatment, and the effect of inhibiting NF-κB transcriptional activity by AH cell line by DHM2EQ was confirmed. It became. On the other hand, luciferase activity was not confirmed in the negative control cell line (K562).

(3) Analysis by confocal microscope

DHM2EQ is thought to inhibit nucleation of subunit p65 of NF-κB. Therefore, ATL cell lines (MT-1, TL-0ml) were treated with 10 µg / ml of DHM2EQ for 24 hours, and the distribution of p65 was examined by confocal microscopy using a fluorescently labeled anti-p65 antibody. In addition, the same study was conducted for each cell line not treated with DHM2EQ.

The results are shown in FIG. In addition, in the figure, "-" shows the result of DHM2EQ non-processing, and "+" shows the result of DHM2EQ process.

As shown in FIG. 7, in the ATL cell lines (MT-1, TL-0ml), the nuclei were detached from black, and nuclear migration of p65 was inhibited by DHM2EQ treatment.

From the results of said (1)-(3), it was confirmed that DHM2EQ inhibits the constitutive NF-κB activation of ATL cells.

Example 5

Inhibitory effect of DHM2EQ on ATL cells

(1) Concentration dependence analysis of proliferation inhibitory effect of DHM2EQ

In 96-well plates, 1 × 10 ⁵ cells / well of each cell of ATL cell line (MT-1, TL-0ml) and control cell line (K562) were dispensed, and the final concentration (2, 5, 10 μg) was determined for DHM2EQ. / ml). An equivalent dose of only solvent, DMSO (0 µg / ml) was used as a control. After 72 hours of incubation, the viability of the cells was determined by MTT assay. The relative MTT value was determined by the ratio of the MTT value of the DHM2EQ treated cells to the MTT value of the DHM2EQ untreated cells, that is, the MTT value of the DHM2EQ treated cells / MTT value of the DHM2EQ untreated cells) × 100 (%). .

The results are shown in FIG. In the drawings, □ indicates K562, ▲ indicates TL-0ml, and ■ indicates the result of MT-1.

As shown in FIG. 8, cell proliferation was inhibited in proportion to the concentration of DHM2EQ in ATL cell lines (MT-1, TL-0ml), whereas cell proliferation was hardly inhibited in control cell line (K562).

(2) Over time analysis of proliferation inhibitory effect of DHM2EQ

The ATL cell line (MT-1, TL-0ml) and the control cell line (K562) were added with DHM2EQ to a final concentration of 10 µg / ml, followed by incubation for 12, 24, 48, and 72 hours. Reviewed by the Essay Act. An equivalent volume of only solvent DMSO was used as a control. Relative MTT value was calculated | required similarly to said (1).

The results are shown in FIG. In the drawings, □ indicates K562, ▲ indicates TL-0ml, and ■ indicates the result of MT-1.

As shown in FIG. 9, cell proliferation was inhibited in proportion to DHM2EQ treatment time in ATL cell lines (MT-1, TL-0ml), whereas cell proliferation was hardly inhibited in control cell line (K562).

(3) Inhibitory effect of DHM2EQ on peripheral blood cells of ATL patients

Monocytes were isolated from ATL patient peripheral blood and ATL cells were isolated. In Patient Case 3, DHM2EQ was added to the isolated ATL cells so as to have a final concentration of 10 µg / ml, and cultured for 24 hours, and the inhibition of proliferation was examined by MTT assay. Proliferation was carried out using the same dose of DMSO as the solvent as a control and the ratio of the MTT value of the DHM2EQ treated cells to the MTT value of the DHM2EQ untreated cells, that is, the MTT value of the DHM2EQ treated cells / MTT value of the DHM2EQ untreated cells. Inhibition was evaluated.

The results are shown in FIG.

As shown in FIG. 10, it was confirmed that DHM2EQ exhibited proliferation inhibitory activity against ATL cells obtained from any ATL patient.

(4) Inhibition of Proliferation of DHM2EQ on Normal Peripheral Blood Monocytes

Monocytes were isolated from normal peripheral blood, DHM2EQ was added at a final concentration of 2, 5, and 10 µg / ml, and cultured for 72 hours, and proliferation inhibition was examined by MTT assay. An equivalent dose of only solvent, DMSO (0 µg / ml) was used as a control. Cell viability was expressed as a ratio with the MTT value of the control (untreated DHM2EQ) as 100%.

The results are shown in FIG.

As shown in FIG. 11, DHM2EQ showed little proliferation inhibitory effect on normal peripheral monocytes.

From the results of said (1)-(4), it was confirmed that DHM2EQ inhibits the proliferation of ATL cells but does not inhibit the proliferation of normal cells.

<Example 6>

Apoptosis Induction of DHM2EQ on ATL Cells

The ATL cell line (MT-1, TL-0ml) and the control cell line (K562) were added with DHM2EQ to a final concentration of 10 µg / ml, followed by 72 hours of incubation. The apoptosis was examined by observing with a microscope. Moreover, the same examination was performed also about adding equal capacity only DMSO which is a solvent.

A result is shown in FIG. 12, In addition, "-" shows the result of DHM2EQ unprocessed (DMSO only), and "+" shows the result of DHM2EQ process.

As shown in FIG. 12, apoptosis was induced by DHM2EQ treatment in ATL cell lines (MT-1, TL-0ml) and fragmentation of nuclei was observed in DHM2EQ untreated cells and control cell lines (K562). Poptosis was not observed.

From these results, it was confirmed that DHM2EQ induces apoptosis of ATL cells but not apoptosis of normal cells.

<Example 7>

Inhibitory Effect of DHM2EQ on Hodgkin's Lymphoma Cells

The time-dependent analysis and the concentration-dependent analysis of the proliferation inhibitory effect of DHM2EQ on Hodgkin's lymphoma cells were carried out in the same manner as in Example 2. As Hodgkin's lymphoma cells, Hodgkin's lymphoma cell lines KMH-2 and L-540 were used.

The results are shown in FIG. In addition, in the drawing, K562, (circle) shows KMH-2, and L shows the result of L-540.

As shown in FIG. 13, cell proliferation was inhibited in proportion to the concentration of DHM2EQ in Hodgkin's lymphoma cell lines (KMH-2 and L-540), whereas cell proliferation was hardly inhibited in the control cell line (K562). In the Hodgkin's lymphoma cell lines (KMH-2, L-540), cell proliferation was inhibited in proportion to the DHM2EQ treatment time, whereas in the control cell line (K562), cell proliferation was hardly inhibited even after elapse of time.

From these results, it was confirmed that DHM2EQ inhibits the proliferation of Hodgkin's lymphoma cells in which NF-κB is constitutively activated, but does not inhibit the proliferation of control cells without NF-κB activation.

<Example 8>

Inhibitory Effect of DHM2EQ on Multiple Myeloma Cells

The concentration-dependent analysis of the proliferation inhibitory effect of DHM2EQ on multiple myeloma cells was performed in the same manner as in Example 2. As multiple myeloma cells, multiple myeloma cell line 196TIB was used.

The results are shown in FIG. In addition, in the figure, "-" shows the result of DHM2EQ non-processing, and "+" shows the result of DHM2EQ process.

As shown in FIG. 14, in the multiple myeloma cell line (196TIB), cell proliferation was inhibited in proportion to the concentration of DHM2EQ.

From this result, it was confirmed that DHM2EQ inhibits the proliferation of multiple myeloma cells.

Example 9

Effect of DHMEQ Inhibiting Binding of NF-κB and DNA in MCF-7 Cells

1. How to

1-1 Preparation of nuclear extract

MCF-7 cells (obtained from Professor Adrian L. Harris, Oxford University) were dispensed at 1 × 10 ⁵ cells / ml in 4 ml aliquots in a 60 mm dish for 2 conditions for one condition. The next day, 2 ml of the medium in a 60 mm dish was collected and treated with DHMEQ adjusted to 1, 3, 10 µg / ml for 2 hours, and then treated with 20 ng / ml of TNF-α. 30 minutes after TNF-α treatment out to pull the medium in 60mm dish in sseokeo (sucker), the cell external use PBS ^- washed twice with in cold PBS ^- into 1ml of, by using the rubber police man (rubber policeman) removing the cells The cell was collected (twice), placed in a 15 ml centrifuge tube, cells were collected from two 60 mm dishes in a 15 ml centrifuge tube, and centrifuged at 1,000 rpm for 5 minutes to remove the supernatant. 700 µl of cold PBS ⁻ was added thereto, the cells were pipetted, collected in a 1.5 ml Eppendorf tube (twice), centrifuged at 3,500 rpm for 5 minutes, and the supernatant was removed. The following operation was performed in ice. It was suspended in 400 µl of buffer A (10 mM HEPES: pH 7.9, 1.5 mM DTT, 0.2 mM PMSF), stirred with vortex, allowed to stand for 15 minutes, and centrifuged at 13,000 rpm for 5 minutes to remove supernatant. In addition, 400 µl of buffer A was added thereto, and the supernatant was removed by centrifugation at 13,000 rpm for 5 minutes. Next, the collected nuclei were suspended in 40 μl of buffer C (20 mM HEPES-KOH: pH7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl ₂ , 0.2 mM EDTA, 0.5 mM DTT, 0.2 mM PMSF), and After standing still for 5 minutes, the mixture was centrifuged at 13,000 rpm for 5 minutes, and the supernatant was collected in eppen to obtain a nuclear extract.

Preparation of 1-2 Probes

4 μl of 1.75 pmol / μl oligonucleotide (Promega: Madison, MA), 2 μl of 10 × T4 PNK buffer (500 mM Tris-HCl: pH8.0, 100 mM MgCl ₂ , 50 mM DTT), and 10 μl of distilled water Furthermore, 2 μl of [ν- ³² P] -ATP and 2 μl of T4 PNK were added and incubated at 37 ° C. for 10 minutes, followed by 80 μl of TE buffer (10 mM Tris-HCl: pH8.0, 1 mM EDTA). Was added to stop the reaction.

Next, the Nick column was placed on a stand, a waste bottle was placed under the column, the caps were removed from the top and bottom, and the TE buffer filled in the column was collected in the waste bottle. About 3 ml of distilled water was added to the column along the wall, and it collect | recovered in the waste bottle. Here, 100 µl of labeled DNA solution was filled in the column without being delivered to the wall. A 1.5 ml Eppendorf tube was prepared below, 400 μl of distilled water was added to the column, and the resulting solution was collected in an Eppendorf tube (fraction 1). A new Eppendorf tube was prepared below, and further, 400 μl of distilled water was filled in the column, and the resulting solution was collected in an Eppendorf tube (fraction 2 = labeled oligonucleotide). After the titration, the cap was closed, and the Geiger counter measured the column, fraction 1, and fraction 2, respectively, and found that fraction 2> column> fraction 1.

In use, the purified labeled DNA probe was diluted with distilled water at about 3 × 10 ⁴ cpm / μl.

The base sequence of a DNA probe is as follows.

5'-ATGTGAGGGGACTTTCCCAGGC-3 '(SEQ ID NO: 3, J. Biol Chem. 277, 24625-24630, 2002)

1-3 binding reaction and gel electrophoresis

4 μl of 5 × binding buffer (375 mM NaCl, 75 mM Tris-HCl: pH7.0, 7.5 mM EDTA, 7.5 mM DTT, 37.5% glycerol, 1.5% NP-40, 1.25 mg / ml BSA), 1 μg / ml poly 1 µl of dI-dC (Amersham Pharmacia Biotech, Inc) and 5 µg of protein were added to the nuclear extract, and water was added so that the total amount was 17 µl. 3 microliters of DNA probes were added thereto, mixed, and incubated at 25 ° C for 20 minutes. Thereafter, 20 µl of the reaction solution was transferred to a well of a 4% polyacrylamide gel and run at 150 V using 0.25 × TBE buffer. After electrophoresis, the gel was dried and photosensitive to the film.

2. Results

In control cells not treated with DHMEQ, bands showing NF-κB and DNA binding were observed 30 minutes after TNF-α treatment, whereas in cells treated with DHMEQ, the binding of NF-κB and DNA was inhibited in a concentration-dependent manner. The binding of NF-κB and DNA was completely inhibited at a concentration of 10 μg / ml (FIG. 15).

Thus, it was shown that DHMEQ inhibits the activation of NF-κB induced by TNF-α.

<Example 10>

In vitro antitumor effect of DHMEQ on human breast cancer cells

Human breast cancer cell line MCF-7 (NF-κB abnormally activating tumor) to DHMEQ; When cultured at 24, 48, 72 hours at 10, 50 µg / ml and the effect of inhibiting growth was examined, 39% at 24 hours, 25% at 48 hours and 17% at 72 hours at 10 µg / ml compared to the control. Cells survived and no viable cells were identified after 24 hours at 50 μg / ml (FIG. 16). Cell proliferation was inhibited depending on concentration and time.

From the above experimental results, DHMEQ suppresses cancer cell proliferation in a time-dependent manner, but it shows that the apoptosis induction effect is small even at a relatively high concentration in a short time, and has a mechanism different from an anticancer agent. This suggests that DHMEQ can be used as a drug with less side effects in vivo.

The detail of the test method is as follows.

MCF-7 was dispensed in 6 well plates at 1 × 10 ⁵ / well and the next day, DHMEQ; The supernatant was transferred to 10, 50 μg / ml medium (5 ml) and control medium (5 ml) only with DHMEQ at the respective concentrations and equivalent amounts of DMSO (used to dissolve DHMEQ). Each group performed 3 wells. The cells were cultured for 24 hours, 48 hours, 72 hours, and then cultured with trypsin + EDTA, and the number of cells was measured with trypan blue to calculate the mean and standard deviation. did. Each of the data was generated and blotted in comparison with control cells only in DMSO.

<Example 11>

In Vivo Antitumor Effect of DHMEQ on Human Breast Cancer Cells

4 mg / kg of DHMEQ was administered to SCID mice (Japanese Crea) subcutaneous tumor model 3 days a week, and the tumor volume was measured over time to examine the effect of inhibiting tumor growth. In addition, the body weight of the mouse was also measured at the same time. As a result, a significant tumor growth inhibitory effect was confirmed (Fig. 17). No death or weight loss of the mice was observed.

The detail of the test method is as follows.

6 x week old SCID mice were inoculated with 1 × 10 ⁶ MCF-7 suspended in 100 μl of PBS. DHMEQ was suspended in 0.5% methyl cellulose solution (methyl cellulose: Nacharitesk) at 200 µl to 4 mg / kg. The drug-containing solution or 0.5% methyl cellulose solution was administered intraperitoneally three times a week from the day after MCF-7 inoculation (8 groups each). Measuring the long diameter and short diameter of the tumor to nogiseu every seven days, and (long diameter) × (minor axis) ^2/2 was calculated tumor volume. At the same time, weight was measured.

As for the so-called anticancer agent, there is a lot of data about the tumor growth inhibitory effect in the animal experiment of the conventional breast cancer therapeutic drug. However, when comparing the efficacy of this drug with a drug having a similar mechanism, an anti-inflammatory COX-2 inhibitor that has become clear in recent years in suppressing tumor growth is reasonable. As a COX-2 inhibitor, there is no report on the effectiveness of MCF-7, but the effect of inhibiting the proliferation of cancer cells that are frequently used in the same manner as other MCF-7 (FIG. 18: Lewis lung tumor, and HT-29 Compared to the COX-2 inhibitor celecoxib (which shows the effectiveness of Pharmacia), it is possible to conclude that there is at least an equivalent effect. The experimental method is as follows (Cancer Res., 60, 1306-1311, March 1, 2000). 10 ⁶ Lewis lung tumors were inoculated into the Fuji of C57 / B16 mice and fed with a mixture of celecoxib (provided by GDSearle / Monsanto Co.) at 160, 480, 1600, and 3200 ppm, respectively, from the day of inoculation. Tumor volume of (n = 20 / group) was measured twice a week using a plethysmometer. 10 ⁶ HT-29 human colorectal cancer cell lines were inoculated into the Fuji of nude mice, orally administered at 160 ppm from the time point when the tumor volume reached 100 mm ³ , and measured once a week. Data is mean ± SD.

As mentioned above, it turned out that DHMEQ has a strong antitumor effect which is independent of apoptosis. Moreover, the low side effect was suggested. Therefore, DHMEQ was found to be effective not only in inhibiting the proliferation of the primary element of breast cancer and in metastasis of cancer from the primary element to other tissues, but also in suppressing the metastasis of the prognostic breast cancer or preventing breast cancer.

At present, the only one used as a preventive agent for breast cancer is the anti-estrogen agent tamoxifen (AstraZeneca) (recognized in North America. In Japan, its usefulness is not confirmed and its use is not approved). The mechanism of action of tamoxifen is to bind to estrogen receptors and antagonize the binding of female hormone estrogen. Therefore, the prevention of carcinogenesis by tamoxifen is limited only to hormone-sensitive breast cancer. In hormonal insensitive breast cancer, carcinogenesis cannot be prevented. Tamoxifen also has side effects that cause uterine body cancer. Other than hormone therapy, there is no indication of breast cancer carcinogenesis. The present drug has a mechanism different from that of the conventional drug having strong cytotoxicity. Therefore, it may be used for cancer prevention in the future. Furthermore, the inactivation of NF-κB by the agent inhibits the production of COX-2, a central mediator of inflammation, upstream of the cascade, similar to NF-κB.

theorem:

1) Since the medicament of the present invention is extremely low in toxicity, it has completely different benefits from anticancer agents.

2) Hormone therapies that target only hormone-sensitive cancers differ greatly in their spectrum. From the above, it is considered clinically and extremely novel.

3) In addition, it is promising as a drug having a novel mechanism from the viewpoint of chemoprevention of breast cancer which attracts attention recently.

<Example 12>

Toxicity test

The acute toxicity test of DHMEQ was performed as follows. DHMEQ was dissolved in 1 drop of 10% DMSO saline + Tween80 and administered to the abdominal cavity of ICR mice, and the life and death of the mice were examined after 24 hours. As a result, 0.156, 0.313, 0.625, 1.25 and 2.5 mg / mouse survived and 5 mg / mouse died immediately. Immediately, the mice that died on the day showed organ deposition and were in small amounts as a result of dissection findings. 5 mg / mouse corresponds to about 250 mg / kg. Acute toxicity LD50 was calculated at 187.5 mg / kg.

Example 13

To assay the effect of the compound represented by the formula (1) or its pharmacologically acceptable salt on cachexia, BALB / c nude mice that induced cachexia were used as model mice of human cachexia patients. At this time, androgen-insensitive human prostate cancer cell line JCA-1 was used to induce cachexia symptoms.

In order to measure the inhibitory effect of NF-κB activity by DHMEQ, NF-κB activity against various concentrations of DHMEQ was measured by using the transcriptional activity of NF-κB. In the reporter, a vector construct (p6kb-Luc) having a luciferase gene as a reporter was used downstream of the promoter having 6 copies of the NF-κB binding sequence. This reporter plasmid was transfected into JCA-1 cells using GenePOETER ™ (Gene Therapy Systems). After 14 hours of transfection, 2.5, 5, 10, 20, and 40 µg / ml of DHMEQ was added to the cell medium, and further cultured for 8 hours, cells were recovered, and luciferase activity was measured. As a control, experiments were conducted simultaneously using nothing treated and one treated only with a solvent containing no DHMEQ (here, DMSO). Each luciferase activity was measured and its absolute value normalized to the respective cell rate. In addition, the whole experiment was performed 3 times independently, and the average value and standard deviation were computed. The results are shown in FIG. 19.

As is clear from FIG. 19, the inhibitory effect of intracellular NF-κB activity increased significantly as the concentration of DHMEQ administered increased. From this, it became clear that DHMEQ inhibits NF-κB activity of JCA-1 cells in a concentration-dependent manner.

<Example 14>

To measure the effect of DHMEQ on the symptoms of cachexia, the nude mice were used to construct cachexia model mice. Subcutaneous 6-week-old nude mice were inoculated with 1 × 10 ⁷ JCA-1 cells suspended in 100 μl of PBS (hereinafter referred to as gallbladder cancer mice). Inoculated mice were randomly divided into three groups at the time of tumor growth to 14 days after inoculation, and group 2 (Gr2; 13 mice) received 8 mg / kg of DHMEQ daily. The group 3 (Gr3; 13) received DMSO daily, and the group 4 (Gr4; 11) received nothing. In addition, normal nude mice not inoculated with JCA-1 cells were designated as Group 1 (Gr1; 14) (hereinafter, referred to as normal mice). From the day after DHMEQ was administered, every other day, tumor weight (FIG. 21) calculated from body weight (FIG. 20) and tumor diameter was measured. On day 26 after the start of DHMEQ administration, the whole mouse was dissected to determine the weight of the tumor (Fig. 22), the weight of fat around the testis (Fig. 23), the weight of the gastrocnemius muscle (Fig. 24), and the hematocrit value (Fig. 25; blood cells). The ratio of the volume to the total blood volume of the component, which can be measured by centrifuging the blood.), And the measured values were aggregated for each group.

The experimental results are described below. First, in the DHMEQ-administered group (Gr2) of 8 mg / kg body weight, the weight loss was significantly suppressed compared to the non-administered groups (Gr3 and Gr4) (FIG. 20). However, at this concentration of DHMEQ, no tumor retraction effect was observed (FIGS. 21 and 22). In the measurement at the end of the experiment (26 days after the start of DHMEQ administration), the weight of fat around the testis (FIG. 23) and the weight of the gastrocnemius muscle (FIG. 24) were determined in the non-administered group (Gr3, Gr4) in the DHMEQ administration group (Gr2). ), The weight loss was significantly suppressed. Moreover, in the hematocrit value, in the DHMEQ-administered group Gr2, there was a significant recovery tendency with respect to the non-administered groups Gr3 and Gr4. At the end of the experiment, the weight of each organ was measured and compared in each group, but an undesirable effect on each organ by administration of DHMEQ was not found (FIG. 26).

As described above, by administering DHMEQ to a mouse that caused cancer by inoculation of JCA-1 cells and caused cachexia symptoms, relief and suppression of cachexia symptoms were prevented even at a concentration that does not affect the size and weight of the cancer. Was observed.

<Example 15>

Inhibitory Effect of DHMEQ on the Constitutive NF-κB Activation of Multiple Myeloma (MM) Cell Lines

2 x 10 ⁶ MM cell lines KMM1, RPMI8226, U266 were treated with 10 μg / ml of DHMEQ for 12 hours, nuclear extracts were prepared from each cell line, and gel shift assays were prepared in the same manner as in Example (1). In addition, the NF-κB inhibitory effect of DHMEQ was examined. As a result, it became clear that the signal by NF-κB disappeared by DHMEQ treatment in MM cell line (FIG. 27A). In addition, TNF treated Jurkat (Jurkat + TNF) was used as a positive control. "Comp" in the figure shows that the signal is specific to the NF-κB binding sequence as a result of the contention inhibition experiment by the unlabeled probe.

Next, the MM cell lines KMM1, RPMI8226, and U266 were treated with 10 µg / ml DHMEQ, nuclear extracts were prepared from each cell line, and gel shift assay was performed in the same manner as in Example (1). The inhibitory effect of NF-κB was examined. The results are shown in FIG. 27B. As a result, it was confirmed that the activation of NF-κB was almost inhibited at 1 hour after DHMEQ treatment, and the inhibition of NF-κB activation was continued even at the time point of 16 hours.

Next, the inhibitory effect of NF-κB was investigated using a reporter assay. First, 6 kB-Luc plasmid DNA was introduced by transfection into MM cell lines KMM1, RPMI8226, U266 (transfected using DMRIE-C (INVITROGEN) on a scale of 2 × 10 ⁵ cells / transfection). ). After 12 hours, treatment was performed with 5 µg / ml DHMEQ for 12 hours, and the transcriptional inhibitory effect of NF-κB by DHMEQ was examined. The results are shown in FIG. 27C. As a result, it became clear that MT-1 and TL-0ml treated with DHMEQ inhibited about 50% of NF-κB transcription, as compared to untreated. Thus, the transcriptional inhibitory effect of NF-κB by DHMEQ appeared.

<Example 16>

Inhibition of Proliferation of Multiple Myeloma (MM) Cell Lines by DHMEQ

In order to investigate the effect of the concentration of DHMEQ on cell proliferation inhibition, the survival rate of the cells was determined by the same method as in Example 5 (1) using the MM cell lines KMM1, RPMI8226, and U266. The result is shown to A of FIG. In addition, the horizontal axis | shaft in a figure shows the density | concentration of DHMEQ, and a vertical axis | shaft shows the relative value of MTT value of the treated cell with respect to DHMEQ untreated cell, ie (DHMEQ processed / untreated) x 100%. As shown in FIG. 28A, it was clear that MM cell lines KMM1, RPMI8226, and U266 inhibit cell proliferation in proportion to the concentration of DHMEQ.

Next, in order to investigate the effects over time of the proliferation inhibition of DHMEQ, by using the MM cell lines KMM1, RPMI8226, U266, by the same method as in Example 5 (2), the proliferation inhibitory effect on the time-lapse Review was conducted by the MTT Essay Act. The results are shown in FIG. 28B. In addition, the horizontal axis in a figure shows the density | concentration of DHMEQ, and a vertical axis | shaft shows the relative value of MTT value of the treated cell with respect to DHMEQ untreated cell, ie, (DHMEQ / untreated) x 100%. As shown in FIG. 28B, it was clear that MM cell lines KMM1, RPMI8226, and U266 inhibit cell proliferation in proportion to the treatment time of DHMEQ.

In addition, in order to investigate the induction of apoptosis in MM cell lines by DHMEQ, MM cell lines KMM1, RPMI8226, and U266 were cultured at concentrations of 10 µg / ml for 0, 24, and 48 hours to obtain annexin V. Apoptosis was examined by dyeing. The result is shown in FIG. Thereby, it became clear that MM cell lines KMM1, RPMI8226, and U266 generate Annexin V positive cells and induce apoptosis by DHMEQ treatment.

In addition, in order to confirm the induction of apoptosis of the MM cell line by DHMEQ, the MM cell lines KMM1, RPMI8226, and U266 were cultured at a concentration of 10 µg / ml for 72 hours, and hex stained to concentrate or fragment the nucleus. Was examined by a fluorescence microscope to investigate apoptosis. The result is shown in FIG. In MM cell lines KMM1, RPMI8226, and U266, fragmented images of nuclei were observed by DHMEQ treatment. From the above results, it was confirmed that DHMEQ induces apoptosis of ATL cells.

<Example 17>

Inhibition of Proliferation of Multiple Myeloma (MM) Patient Cells by DHMEQ

Next, in order to investigate the proliferation inhibitory effect of DHMEQ's multiple myeloma (MM) patient cells, MM cells (MM1, MM2, MM3) were isolated from the patient's bone marrow, and cultured for 48 hours at a concentration of 10 µg / ml of DHMEQ. Inhibition of growth was examined by the MTT assay. The result is shown to A of FIG. In addition, monocytes (PBMC) isolated from normal peripheral blood were used as controls. The vertical axis in the figure represents the relative value of the MTT value of the treated cells to the DHMEQ untreated cells, that is, (DHMEQ untreated / untreated) × 100%. As shown in FIG. 29A, the proliferation of MM cells from patients with multiple myeloma (MM) was suppressed in proportion to the concentration of DHMEQ, and the effect on monocytes isolated from normal peripheral blood was hardly confirmed.

From this, it became clear that DHMEQ has a proliferative inhibitory activity against MM cells of MM patients, but has little effect on normal monocytes and can function as a pharmaceutical composition with fewer side effects.

In addition, in order to investigate the induction of apoptosis in multiple myeloma (MM) patient cells by DHMEQ, 72 hours of incubation was performed on the cells of multiple myeloma (MM) at a concentration of 10 µg / ml of DHMEQ and hex. Examination of apoptosis was carried out by tho dyeing and observing the concentration or fragmentation of the nucleus with a fluorescence microscope. The result is shown in FIG. As shown in FIG. 29B, MM cells from multiple myeloma (MM) patients were confirmed to have apoptosis induced by DHMEQ treatment and nucleus fragmentation.

Example 18

Inhibitory Effect of DHMEQ on Constitutive NF-κB Activation of Hodgkin's Lymphoma (HL) Cell Line

2 × 10 ⁶ HL cell lines KMH2, L428, L540, HDLM2 and control cell line K562 were treated with 10 μg / ml of DHMEQ for 12 hours, and nuclear extracts were prepared from each cell line, the same method as in (1) of Example 4 Gel shift assays were conducted to examine NF-κB inhibitory activity. As a result, it became clear that the HL cell line almost lost NF-κB signal by DHMEQ (FIG. 30A). Cell line K562 (myeloid cell leukemia cell line) is a cell line that does not recognize constitutive activation of NF-κB and was used as negative control. On the other hand, TNF treated Jurkat (Jurkat + TNF) was used as a positive control. "Comp" in the figure indicates that the signal is specific for the NF-κB binding sequence in the contention inhibition experiment by the unlabeled probe.

Next, the HL cell lines KMH2 and L540 were treated with 10 µg / ml DHMEQ, nuclear extracts were prepared from each cell line, and gel shift assay was carried out by the same method as in Example (1), followed by time course. The inhibitory effect of NF-κB was examined. As a result, it was confirmed that the activation of NF-κB was almost inhibited at 1 hour after the treatment, and the inhibition of NF-κB activation was continued even at the time point of 16 hours (FIG. 30B).

NF-κB is a complex consisting of constituents of p50, p65 and c-Rel. Therefore, the constitutive factors of NF-κB that are constitutively activated in the HL cell lines KMH2, L428, L-540, and HDLM2 were examined. In the same manner as in Example 1, supershifting was performed using antibodies against p50, p65, and c-Rel of NF-κB. The result is shown to FIG. 30C. As shown in FIG. 30C, supershift was observed especially for p50, and it was confirmed that p50 was present in the complex in any of the HL cell lines.

In L428 and KMH2, the weak signal remains after the DHMEQ treatment. Thus, we examined whether these signals had specific constituents of NF-κB that were DHMEQ resistant. In addition, the HL cell lines KMH2, L428L-540, and HDLM2 were treated with 5 μg / ml of DHMEQ for 12 hours, and the remaining components of NF-κB were treated with antibodies against p50, p65, and c-Rel of NF-κB. Super shift was performed similarly to the method described in Example 1. The result is shown in FIG. As shown in D of FIG. 30, the remaining components were mainly obtained at the same result as p of FIG. 30 at p50, and it was considered that there was no characteristic constitution factor showing resistance to DHMEQ.                 

Example 19

Enhancement of antitumor agent effect by DHMEQ

The antitumor activity enhancement effect by DHMEQ was examined using camptothecin (CPT), daunomycin (DNR), and etoposide (ETP) as antitumor agents. Antitumor action and its enhancement effect were examined by the MTT assay described in (2) of Example 5. The result is shown in FIG. In addition, in this Example, the cell used KMH2. The horizontal axis in the figure shows the concentration of each antitumor agent and the DHMEQ concentration. The concentration of anticancer drugs was three steps, respectively, and only DHMEQ and only antitumor drugs were examined for each use. DHMEQ was used at a concentration of 10 µg / ml, and the treatment time was 48 hours. The vertical axis represents the relative value of the MTT value of the treated cells to the untreated cells, that is, (treated / untreated) × 100%. 31A used camptothecin (CPT) as an antitumor agent, B was daunomycin (DNR) as an anticancer agent, and C was etoposide (ETP) as an antitumor agent. As a result, it was shown that DHMEQ enhances the effect of antitumor agent in all.

Example 20

Antitumor potentiation effect by DHMEQ is due to inhibition of NF-κB activation by antitumor agent.

NF when tumor cells were treated with antitumor agents (camptothecin (CPT), daunomycin (DNR), etoposide (ETP)) to investigate NF-κB activation by each antitumor agent κB activation was examined by the gelshift assay described in (1) of Example 4. The result is shown in FIG. In addition, in this Example, the cell used KMH2. The lower panel of each panel in the figure quantifies the obtained signal and shows the relative value when 1 is set before the treatment. As shown in FIG. 32A, it was shown that even when treated with any anti-tumor agent, 3 to 20-fold NF-κB activity was generated in tumor cells on a daily basis, compared with that before treatment.

In tumor cells, to examine the constitutive factors of NF-κB induced by camptothecin (CPT) and daunomycin (DNR), p50, p65, and c- of NF-κB are the same as those described in Example 1. Supershift was performed using an antibody against Rel. The result is shown in FIG. As shown in B of FIG. 32, it was clear that activated NF-κB mainly contained p50 in tumor cells treated with camptothecin (CPT) or daunomycin (DNR).

Next, in order to investigate the inhibition of NF-κB activation by antitumor agents by DHMEQ, tumor cells were treated by using DHMEQ in combination with each antitumor agent (camptothecin (CPT), daunomycin (DNR)). The gel shift assay described in (1) of Example 4 was performed for the induction of NF-κB at one time, and the effect of NF-κB inhibition over time was examined. The result is shown to C of FIG. In addition, in this Example, the cells used KMH2. The lower panel of each panel in the figure quantifies the obtained signal and shows the relative value when 1 is set before the treatment. As shown in FIG. 32C, NF-κB induction was strongly inhibited by DHMEQ in combination with any antitumor agent.

Example 21

SCID mice inoculated with ATL cell line intraperitoneally were used to investigate the effect of DHMEQ in vivo. First, SCID (Male C. B17-scid / scid scid / scid) mice (CB17 strain, 5 weeks old, male; SLC Japan, Inc (Shizuoka, Japan)) were injected with 1 mg of IL-2 receptor antibody (TM-β1; J). 147: 2222-2228, 1991) for 3 to 5 days and 3-4 × 10 ⁷ ATL cell line MT-2 was inoculated intraperitoneally. Thereafter, these mice were administered with DHMEQ 4 mg / kg-weight or 12 mg / kg-weight dissolved in 0.5% CMC (carboxymethyl cellulose; Sigma) solution three times a week intraperitoneally, and the survival rate and the condition of the mice were observed. did. In the control group, 0.5% CMC solution without DHMEQ was administered intraperitoneally three times a week over the same month. The result is shown in FIG. In addition, the survival curve was calculated by the Kaplan and Myer method, statistical significance was determined by the Cox-Mantel test. As shown in Figure 33, the DHMEQ 4mg / kg administration group (DHMEQ (+)) after the start of the experiment, about 4 days out of 6 survived, and the control group (DHMEQ (-)) was five cases ( Death was complete and statistically significant difference was obtained (Cox-Mantel test; p <0.05). The DHMEQ 12 mg / kg administration group (DHMEQ (+)) survived 5 out of 6 animals about 30 days after the start of the experiment, whereas the non-administered group (DHMEQ (-)) died of 4 mice. Statistically significant differences were obtained (Cox-Mantel test; p <0.05). In addition, the administration group (DHMEQ (+)) was not confirmed abnormal in the body weight of the mouse, and toxicity was not confirmed even when using 3 times the amount of DHMEQ 4mg / kg. As a result, it became clear that DHMEQ can rescue the death of mouse individuals by transplanted ATL cells in vivo.

<Example 22>

Synergistic effect of using DHMEQ and radiation

The effects of DHMEQ on the proliferation of tumor cells against human pancreatic cancer in vivo were investigated. 2 x 10 ⁶ human pancreatic cancer cell line PK-2 was inoculated subcutaneously in the right dorsal area of SCID mice (7 mice) and intraperitoneally injected (ip) with DHMEQ 12 mg / kg once daily at 200 μl for 5 days. After that, the long and short diameters of the tumor were measured every 7 days, and the tumor volume was calculated by (long diameter) × (short diameter) ² × 0.5. The results are shown in FIG. As shown in FIG. 34, it became clear that the proliferation of tumor cells was significantly inhibited by using DHMEQ.

Next, the apoptosis enhancing effect of DHMEQ on the irradiated tumor cells was investigated. Human pancreatic cancer cell line Colo357 treated with 10 μg / ml of DHMEQ for 6 hours and Colo357 incubated for 6 hours by irradiation of 20 Gy and human pancreatic cancer cell line Colo357 incubated for 6 hours with 20 Gy of radiation The cell line Colo357 was double-stained with Annexin-V and PI (propidium iodide), respectively, and the ratio of the early stages of apoptosis was measured by flow cytometry. The result is shown in FIG. As shown in FIG. 35, DHMEQ alone did not induce apoptosis in human cancer cell line Colo357. On the other hand, it was clear that after 20 Gy irradiation, the cells to which DHMEQ was administered increased about three times the induction of apoptosis, compared with the cells which only irradiated 20 Gy.                 

In addition, we investigated the effects of in vitro proliferation inhibition on human cancer cell lines in combination with DHMEQ radiation. 5 × 10 ⁵ human cancer cell lines Panc-1, PK-8, and Colo357 were irradiated with 2.5 Gy or 10 Gy after 10 days in a 10 cm dish, followed by 4 hours of 10 μg / ml DHMEQ in the combination group. The cell number was measured after 24 hours. In addition, 0 hour after completion | finish of irradiation or the addition of a chemical agent was made. The result is shown in FIG. As a result, DHMEQ had sufficient proliferation inhibitory effect against human cancer cell lines PK-8 and Colo357 with only 4 hours of drug contact, and the effect was comparable to irradiation of 2.5Gy equivalent. In Panc-1, in which the effect of DHMEQ alone was poor on radiation resistance, treatment resistance was lost in combination with irradiation and DHMEQ. In Colo357 and Panc-1, in combination with irradiation, DHMEQ showed synergistic antiproliferative effect, and it was clear that the 2.5Gy irradiation effect was increased to 4 times 10Gy equivalent.

According to the present invention, it is possible to provide a pharmaceutical composition that can improve the symptoms accompanying the activation of NF-κB.

<110> KEIO UNIVERSITY <120> Pharmaceutical composition comprising NF-kappaB inhibitor <130> PCT / 628 <150> JP02 / 185866 <151> 2002-06-26 <150> JP03 / 37167 <151> 2003-02-14 <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> synthetic DNA <400> 1 agttgagggg actttcccag gc 22 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> synthetic DNA <400> 2 tcaactcccc tgaaagggtc cg 22 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> synthetic DNA <400> 3 atgtgagggg actttcccag gc 22  

Claims (46)

A pharmaceutical composition containing a compound represented by the following formula (1) or a pharmacologically acceptable salt thereof as an active ingredient for improving at least one symptom caused by tumor cells. [Formula 1]

(In formula, R <^1> is a hydrogen atom or a C2-C4 alkanoyl group, and R <^2> is group represented by either of following formula (A)-(G).)

(In formula, R <^3> is a C1-4 alkyl group.) The method of claim 1, A pharmaceutical composition, characterized by amelioration of at least one symptom by apoptosis of the tumor cells. The method of claim 1, A pharmaceutical composition, characterized in that the apoptosis of the tumor cells does not contribute and ameliorates at least one symptom caused by the tumor cells. The method of claim 3, wherein A pharmaceutical composition, characterized by amelioration of at least one symptom caused by the tumor cells by inhibiting the activation of NF-κB. The method of claim 3, wherein A pharmaceutical composition, wherein said symptom is a tumor metastasis. The method of claim 5, wherein A pharmaceutical composition characterized by improving tumor metastasis by inhibiting adhesion with vascular endothelial cells. The method of claim 1, By inhibiting the proliferation of the tumor cells, the pharmaceutical composition, characterized in that to improve at least one symptom caused by the tumor cells. The method of claim 1, The pharmaceutical composition, characterized in that the symptom is selected from the group consisting of Hodgkin's disease, cancer cachexia, leukemia. The method of claim 3, wherein A pharmaceutical composition, wherein the tumor cells are breast cancer cells. The method of claim 1, A pharmaceutical composition, wherein the compound is the following formula (1a) or (1b).

The method of claim 8, A pharmaceutical composition, characterized by preventing or ameliorating at least one symptom of weight loss, reduction of hematocrit, reduction of fat, and reduction of muscle, which are symptoms of the cancer cachexia. The method of claim 3, wherein By inhibiting angiogenesis in the tumor formed by the tumor cells, a pharmaceutical composition, characterized in that to improve at least one symptom caused by the tumor cells. By inhibiting the activation of NF-κB by a treatment that activates NF-κB, the compound represented by the following formula (1) or a pharmacologically acceptable salt thereof, which can enhance the effect of the treatment, is contained as an active ingredient. Pharmaceutical composition to say. [Formula 1]

(In formula, R <^1> is a hydrogen atom or a C2-C4 alkanoyl group, and R <^2> is group represented by either of following formula (A)-(G).)

(Wherein, R ³ is a C 1-4 alkyl group.) The method of claim 13, The pharmaceutical composition characterized in that the treatment for activating the NF-κB is treatment using an anti-tumor agent. The method of claim 13, The pharmaceutical composition characterized in that the treatment for activating the NF-κB is treatment by radiation to tumor cells. The method of claim 14, A pharmaceutical composition comprising the antitumor agent as an active ingredient. The method of claim 14, The anti-tumor agent is a camptothecin, or a drug composition, characterized in that it is daunobicin. The method of claim 13, A pharmaceutical composition, wherein the compound is the following formula (1a) or (1b).

A tumor cell proliferation inhibitor for inhibiting the proliferation of tumor cells containing the compound represented by the following formula (1) or a pharmacologically acceptable salt thereof as an active ingredient. [Formula 1]

(In formula, R <^1> is a hydrogen atom or a C2-C4 alkanoyl group, and R <^2> is group represented by either of following formula (A)-(G).)

(In formula, R <^3> is a C1-4 alkyl group.) The method of claim 19, Tumor cell proliferation inhibitor, characterized in that the compound is the following formula (1a) or (1b).

An adhesion molecule expression inhibitor for inhibiting the expression of adhesion molecules of vascular endothelial cells, which contains the compound represented by the following formula (1) or a pharmacologically acceptable salt thereof as an active ingredient. [Formula 1]

(In formula, R <^1> is a hydrogen atom or a C2-C4 alkanoyl group, and R <^2> is group represented by either of following formula (A)-(G).)

(In formula, R <^3> is a C1-4 alkyl group.) The method of claim 21, Adhesive compound expression inhibitory agent derived from vascular endothelial cell, characterized in that the compound is the following formula (1a) or (1b).

An apoptosis inducer for inducing apoptosis of tumor cells, which contains a compound represented by the following formula (1) or a pharmacologically acceptable salt thereof as an active ingredient. [Formula 1]

(In formula, R <^1> is a hydrogen atom or a C2-C4 alkanoyl group, and R <^2> is group represented by either of following formula (A)-(G).)

(In formula, R <^3> is a C1-4 alkyl group.) The method of claim 23, The apoptosis inducer, wherein the compound is the following formula (1a) or (1b).

An agent for preventing and treating arteriosclerosis, which contains a compound having an NF-κB inhibitory activity as an active ingredient. The method of claim 25, A prophylactic and therapeutic agent, wherein the compound having an NF-κB inhibitory effect is a compound represented by the following formula (1) or a pharmacologically acceptable salt thereof. [Formula 1]

(In formula, R <^1> is a hydrogen atom or a C2-C4 alkanoyl group, and R <^2> is group represented by either of following formula (A)-(G).)

(In formula, R <^3> is a C1-4 alkyl group.) A prophylactic and therapeutic agent for cancer containing a compound having an NF-κB inhibitory activity as an active ingredient. The method of claim 27, A prophylactic and therapeutic agent, wherein the compound having an NF-κB inhibitory effect is a compound represented by the following formula (1) or a pharmacologically acceptable salt thereof. [Formula 1]

(In formula, R <^1> is a hydrogen atom or a C2-C4 alkanoyl group, and R <^2> is group represented by either of following formula (A)-(G).)

(In formula, R <^3> is a C1-4 alkyl group.) The method of claim 27, A prophylactic and therapeutic agent used to suppress metastasis of cancer. A cachexia therapeutic agent containing a compound represented by the following formula (1) or a pharmacologically acceptable salt thereof as an active ingredient. [Formula 1]

(In formula, R <^1> is a hydrogen atom or a C2-C4 alkanoyl group, and R <^2> is group represented by either of following formula (A)-(G).)

(In formula, R <^3> is a C1-4 alkyl group.) The method of claim 30, The cachexia therapeutic agent, wherein the compound is the following formula (1a) or (1b).

The method of claim 30, A cachexia therapeutic agent, which is a therapeutic agent for cancer cachexia in a tumor patient. The method of claim 30, A cachexia therapeutic agent which prevents or improves at least one symptom of weight loss, decrease in hematocrit value, decrease in fat, and decrease in muscle, which are symptoms of cancer cachexia in a tumor patient. A cachexia therapeutic agent containing a compound having an NF-κB inhibitory effect as an active ingredient. A method of treatment comprising using a compound represented by the following formula (1) or a pharmacologically acceptable salt thereof for ameliorating at least one symptom caused by tumor cells. [Formula 1]

(In formula, R <^1> is a hydrogen atom or a C2-C4 alkanoyl group, and R <^2> is group represented by either of following formula (A)-(G).)

(In formula, R <^3> is a C1-4 alkyl group.) 36. The method of claim 35 wherein Treatment by the apoptosis of the tumor cells, characterized in that to improve at least one symptom. 36. The method of claim 35 wherein And the apoptosis of the tumor cells does not contribute and ameliorates at least one symptom caused by the tumor cells. 36. The method of claim 35 wherein Inhibiting the activation of NF-κB, thereby improving at least one symptom caused by the tumor cells. 36. The method of claim 35 wherein And said symptom is a symptom selected from the group consisting of tumor metastasis, a symptom resulting from proliferation of said tumor cells, Hodgkin's disease, and cachexia. 36. The method of claim 35 wherein The compound is a therapeutic method characterized by the following formula (1a) or (1b).

A method of treatment comprising using a compound represented by the following formula (1) or a pharmacologically acceptable salt thereof for improving arteriosclerosis by inhibiting adhesion between vascular endothelial cells and leukocytes. [Formula 1]

(In formula, R <^1> is a hydrogen atom or a C2-C4 alkanoyl group, and R <^2> is group represented by either of following formula (A)-(G).)

(In formula, R <^3> is a C1-4 alkyl group.) 42. The method of claim 41 wherein The compound is a therapeutic method characterized by the following formula (1a) or (1b).

And a step of performing a treatment for activating NF-κB and administering a pharmaceutical composition containing a compound represented by the following formula (1) or a pharmacologically acceptable salt thereof as an active ingredient. . [Formula 1]

(In formula, R <^1> is a hydrogen atom or a C2-C4 alkanoyl group, and R <^2> is group represented by either of following formula (A)-(G).)

(In formula, R <^3> is a C1-4 alkyl group.) The method of claim 43, The treatment which activates said NF-κB is administration of an anti-tumor agent. The method of claim 43, The treatment which activates said NF-κB is a treatment by radiation to a tumor cell. The method of claim 43, The compound is a therapeutic method characterized by the following formula (1a) or (1b).

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